Immunogenic complex

ABSTRACT

The invention relates to an immunogenic complex comprising at a least one glycoside and at least one lipid, integrated into an iscom complex or matrix, and at least one antigen which antigen is integrated into the iscom complex or coupled on to or mixed with the iscom complex or iscom matrix complex, characterised in that it also comprises at least one enzyme. It also relates to such a complex further comprising at least one peptide which specifically binds to a receptor expressed on a cell capable of antigen presentation, which cell expresses MHC Class I or Class II and to compositions comprising the complexes.

[0001] The present invention relates to new immunogenic complexes of iscom or iscom matrix and antigens together with an ene and possibly also peptides, which specifically binds to a receptor expressed on a cell capable of antigen presentation, and to compositions comprising the new complexes.

BACKGROUND OF THE INVENTION

[0002] Exploitation of the mucosal immune system offers several advantages from a vaccine point of view. Mucosal vaccines may achieve both systemic and local mucosal immune protection against infectious micro-organisms of which many gain access to the body via mucosal membranes. There is a growing interest for oral vaccines and for the possibility of using such vaccines to protect against infectious diseases affecting not only mucosal surfaces but also against diseases like HIV, polio etc.

[0003] Mucosally active vaccines containing recombinant protein antigens would have many immunological and economic advantages for inducing protective immunity against a wide variety of mucosal and systemic pathogens.

[0004] Recent results show that antigens incorporated into iscoms are highly immunogenic by mucosal routes, inducing strong T cell mediated immune responses that include secretory IgA antibodies, Th1 dependent delayed type hypersensitivity (DTH) and cytokine production, as well as very strong class I MHC restricted CD8⁺ T cell responses. Serum antibody production is also primed, but this is relatively less efficient (3, 11).

[0005] The particulate nature of iscoms allows them to preferentially target and activate accessory cells such as macrophages and dendritic cells (DC) (14-17), Iscoms induce normal responses in IL4KO, but not in IL12KO mice (3, 12 and 18). In addition, iscoms stimulate the production of mediators such as IL1, IL6 and IL12 from macrophages and DC and in vivo depletion of macrophages markedly reduces the adjuvant effects of iscoms (15, 18-22).

[0006] There is a group of bacterial toxins that exert strong enzymatic activity on mammalian cells, such as E. coli heat-labile toxin (LT) and choler toxin (CT). They act by ADP-ribosylation of GTP-binding proteins in the cell membrane of the target cells, resulting evenly in the formation of large quantities of intracellular cAMP. The increase in cAMP may then act to immunomodulate many diverse immune reactions such as increasing B lymphocyte differentiation, augmenting co-stimulation of antigen-presenting cells, inhibiting or promoting various T cell functions or modulating apoptotis in lymphoid cells. They are therefore potent adjuvants.

[0007] CT is composed of five enzymatically inactive, non-toxic B-subunits (CTB) held together in a pentamere structure surrounding a single A-subunit that contains a linker to the pentamere via the A2 fragment (CTA2) and the toxic enzymatically active A1-fragment (CTA1) of the molecule.

[0008] The toxic CTA1 bas strong ADP-ribosyl transferase activity. This results in activation of adenylate cyclase and the subsequent intracellular increase in cAMP.

[0009] CTB binds to the ganglioside GM1-receptor, present on most mammalian cells including lymphocytes and gut epithelial cells. CTB has been integrated in ISCOMS as a mucosa targeting molecule EP 97905539.9 and also together with antigens that do not easily penetrate mucosas EP 97905541.5, in order to direct orally administrated iscoms to the mucosa.

[0010] Although it has been shown that CT is a potent inducer of most T cell dependent responses when given orally (3, 4), it has also been reported that it may be less efficient at stimulating CD4⁺ Th1 cells than Th2 cells (5-7). Furthermore, it is not widely accepted as being able to prime CD8⁺ T cells, while the toxicity of intact CT is likely to prevent its use as a practical vaccine vector in man. Recently attempts have been made to overcome this problem by using an artificial adjuvant vector composed of the enzymatically active A1 fragment of CT (CTA1) linked to two Ig binding domains of staphylococcal protein A. The resulting CTA1-DD fusion protein binds B lymphocytes specifically, has no systemic toxicity and has similar adjuvant properties to CT holotoxin when given by parenteral routes (8)+. However, preliminary indications are that it may have only limited effects when given orally.

[0011] Whereas B lymphocytes play a central role in the adjuvant effects of CT and in particular, CTA1-DD (8, 13), the particulate nature of iscoms allows them to preferentially target and activate accessory cells such as macrophages and dendritic cells (DC) (14-17). Thus, the mucosal adjuvant effects of CT are dependent on the presence of IL4 dependent B cell follicles in Peyer's patches (PP), but not on IL12 (3, 13). In contrast, iscoms show an opposite pattern of requirements, inducing normal responses in IL4KO, but not in IL12KO mice (3, 17, and 18). In addition whereas CT may inhibit many functions of macrophages, iscoms stimulate the production of mediators such as IL1, IL6 and IL12 from macrophages and DC and in vivo depletion of macrophages markedly reduces the adjuvant effects of iscoms (15, 18-22).

[0012] Thus, iscoms and CTA1 and its derivatives use different anatomical routes and immune mechanisms to induce mucosal immune responses.

SUMMARY OF THE INVENTION

[0013] It has now turned out that when combining iscoms and an enzyme, especially CTA1 and its derivatives, their adjuvant effects are enhanced, some of their limitations and disadvantages are overcome and that the over all effect unexpectedly may be synergistic. Surprisingly the enzymatic activity of CTA1 is kept intact in the complex. This novel formulation is non-toxic and is highly immuogenic by a variety of mucosal and systemic routes.

[0014] The main object of the invention is to provide an immunogenic complex comprising at least one glycoside, at least one lipid and at least one antigen which antigen is integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex, characterized in that it also comprises at least one enzyme.

[0015] Another object of the invention is to provide immunogenic iscom complexes, comprising at least one glycoside, at least one lipid and at least one antigen, into which an enzyme preferably A1 subunits of a bacterial enterotoxin have been integrated.

[0016] Another object is to provide immunogenic iscom complexes into which both enzymes and peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation, have been integrated.

[0017] Another object is to provide iscom complexes on to which antigens and enzymes and/or peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation, have been coupled.

[0018] Another object is to provide iscom complexes mixed with antigens and enzymes and/or peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation.

[0019] Another object of the invention is to provide immunogenic iscom matrix complexes, comprising at least one glycoside and at least one lipid on to which antigens, enzymes and/or peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation have been coupled.

[0020] Another object is to provide iscom matrix complexes mixed with antigens and enzymes and/or peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation.

[0021] Still another object is to provide a complex where the enzyme and a peptide or protein which specifically binds to a receptor is bound together into a fusion protein which is integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex.

[0022] Still another object is to provide a complex where the enzyme, the peptide or protein which specifically binds to a receptor and an antigen is bound together into a fusion protein, which is integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex.

[0023] Another object is to provide a composition comprising the new complexes according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The invention relates to a immunogenic complex comprising at least one glycoside, at least one lipid and at least one antigen which antigen is integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex, characterized in that it also comprises an enzyme.

[0025] The enzyme is preferably an enzyme that confers enzymatic ADP-ribosylating activity as it has turned out that such an enzyme has unexpected adjuvant activity in combination with iscom and iscom matrix complexes. If the enzyme is toxic it is preferred that the toxic put be deleted from the enzyme. Thus, the enzyme may be an enzyme with ADP-ribosylating activity from which a toxic part has been deleted. Especially the enzyme is a native or mutant bacterial toxin, preferably an enterotoxin and specifically a subunit of a toxin that confers enzymatic ADP-ribosylating activity. The enzyme may be selected from Cholera toxin (CT), E. Coli heat labile enterotoxin (LT), Pertussis, Clostridia, Shigella and Peudomonas toxins. Most preferably the enzyme is at least one A1 subunit of a bacterial enterotoxin wherein said enterotoxin is selected from the group consisting of cholera toxin (CT) and E. Coli heat labile enterotoxin (LT). Such enzymes and subunits and the production thereof are described in U.S. Pat. No. 5,917,026.

[0026] According to a preferred form of the invention, the immunogenic complex further comprises at least one peptide or protein, which specifically binds to a receptor expressed on a cell capable of antigen presentation. Preferably the cell expresses MHC Class I or Class II antigen. The antigen-presenting cell may be belong the group consisting of lymphocytes, macrophages, dendritic cells, Langerhans cells and epithelial cells.

[0027] Iscom contains at least one glycoside, at least one lipid and at least one type of antigenic substances, especially proteins and peptides and can be produced as described in EP 0 109 942 B1, EP 0 242 380 B1 and EP 0 180 564 B1.

[0028] Iscom matrices contain at least one glycoside and at least one lipids. Matrices have an is immunostimulating effect on administration together with antigenic substances, and can be produced as described in EP 0 436 620 B1.

[0029] The enzyme and/or the antigen and/or the peptide or protein which specifically binds to a receptor may be integrated into an iscom complex. It is also possible to couple one or mote of these substances an to an iscom complex already containing antigens or on to an iscom matrix complex.

[0030] Further is it possible to mix on or more of these substances with iscom or iscom matrices. In such a case the iscom complex may already contain one or more antigens and/or one or more enzymes and/or one or more peptides or proteins, which specifically bind to a receptor.

[0031] Thus, the invention relates to a composition comprising iscoms wherein one or more antigens, one or more enzymes or one or more receptor binding peptides or proteins are integrated into, coupled on to or mixed with the iscom complex.

[0032] The invention also relates to a composition comprising matrix and one or more antigens, one or more enzymes and one or more receptor binding peptides or proteins coupled on to or mixed with the matrix complex.

[0033] The antigen-presenting cells having receptors to which the peptide can bind, are suitably cells capable of antigen presentation especially cells expressing MHC Class I and Class II and may be lymphocytes, such as B-lymphocytes, T-cells, monocytes, macrophages, dendritic cells, Langerhans cells, and epithelial a endothelial cells.

[0034] The peptide is a peptide that binds to receptors of the above cells, preferably to an Ig or Fc receptor expressed by said antigen-presenting cell and most preferably to receptors of B-lymphocytes.

[0035] Examples of specific targeting peptides are peptides capable of binding to receptors of:

[0036] (i) granulocyte-macrophage colony-stimulating factor (GM-CSF) capable of binding to the GM-CSF receptor .alpha./.beta.heterodimer present on monocytes, neutrophils, eosinophils, fibroblasts and endothelial cells,

[0037] (ii) CD4 and CD8 expressed on T cells which together with the T cell receptor (TcR) act as co-receptors for MHC class II and MHC class I molecules, respectively, MHC class I are expressed on most nucleated cells, whereas MHC class II molecules are expressed on dendritic cells, B cells, monocytes, macrophages, myeloid and erythroid precursor cells and some epithelial cells,

[0038] (iii) CD 28 and CTLA-4, two homodimeric proteins expressed mainly on T cells which bind to B7 expressed on B cells,

[0039] (iiii) CD40 present mainly on the surface of mature B cells which interact with gp39 expressed on T cells,

[0040] (iiiii) different isotypes of the Ig heavy chain constant regions which interact with a number of high or low affinity Fc receptors present on mast cells, basophils, easinophils, platelets, dendritic cells, macrophages, NK cells and B cells.

[0041] According to a particularly preferred embodiment of the invention, said peptide is constituted by protein A or a fragment thereof in single or multiple copies, such as one or more D subunits thereof.

[0042] According to the invention, the enzyme and the peptide which specifically binds to a receptor may be bound together into a fusion protein, which may be integrated into au iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex.

[0043] The fusion proteins comprise a sub-unit of a native or mutant bacterial toxin that confers enzymatic ADP-ribosylating activity, and, lined thereto, a peptide. The peptide is preferably such that the resulting fusion protein is in possession of water solubility and capability of targeting the fusion protein to a specific cell receptor different from receptors binding to the native toxin; thereby mediating intracellular uptake of at least said subunit.

[0044] An antigen may also be incorporated in the fusion protein fusion, the antigen, the receptor binding peptide or protein and the enzyme may be used as a single molecule or as different combinations in fusion proteins for integration into iscoms or coupling on to iscoms and/or matrices or mixing iscoms and/or matrices. One or more antigens, one or more receptor binding peptides or proteins and one or more enzymes may be used as single molecules or in the fusion protein.

[0045] The integration of the substances and the coupling thereof on to iscoms or iscom matrices maybe done as described in EP 0 109 942 B1, EP 0 242 380 B1 and 0 180 564 B1. Although the invention is by no means limited hereto it will be exemplified in the following mainly with reference to the sub-unit A1 of cholera toxin or a mutant thereof.

[0046] Preferably the fusion protein comprises the A1 subunit of cholera toxic and is fused to one or more copies of protein A or a fragment thereof, such as the D region of said protein A.

[0047] One fusion protein denoted CTA1-DD consisting of CTA1 linked to DD, a dimer of the D-region of protein A, binds to soluble immunoglobulins as well as the Ig-receptor on B cells. The results demonstrate that is molecule lacks enterotoxic activity, but so effectively ADP-ribosylates target protein. When used as a parenteral adjuvant CTA1-DD enhances anti-KLH antibody responses and increases KLH T cell priming.

[0048] These results demonstrate the possibility to circumvent the toxic effects of CT simply by removing the CTB pentamer, thus excluding the potential interaction resulting in toxicity between the epithelial cell GM1-receptor and CT. The strategy of targeting of the immunomodulating activity of CTA1 to defined cell populations can be expanded to include essentially any given cell type, enabling specific modulation of cellular responses controlled by cAMP, provided that a suitable targeting molecule is available. CTA1 alone is highly insoluble in physiological aqueous solutions. Thus, the targeting molecule used as fusion partner in this invention also has the important function to enhance solubility of the CTA1 entity.

[0049] The CTA1 moiety in CTA1-Dr) is targeted to B cells primarily, and away from the GM1-receptor on e.g. the gut epithelial cells. Furthermore, using this construct it has been demonstrated that

[0050] (i) the enzymatic activity of CTA1 was retained in CTA1-fusion proteins provided that CTA1 was fused at its carboxy terminus;

[0051] (ii) CTA1 in the fusion protein exerts its ADP-ribosyltransferase activity in target cells through a pathway for entry that is different from the surface ganglioside GM1-receptor; and that.

[0052] (iii) CTA1-DD displays a strong immunopotentiating activity.

[0053] Similarly, CTA1 may be fused to other targeting molecules such as e.g. CD4 to access MHC II expressing cells or any other ligand that specifically can bind to a receptor present on the cell surface. Using this approach CTA1 will not interact with the GM1-receptor present on most mammalian cells including gut epithelial cells because the CTB portion is lacking in the construct. There fore, CTA1 is given a narrow spectrum of cellular interactions via specific binding to surface Ig or Fc-receptors thereby targeting CTA1 to primary B cells, and macrophages and other Fc-receptor carrying cells.

[0054] Fusion proteins may be produced by general biotechnological methods known in the art. Fusion protein CTA1DD may be produced as described in U.S. Pat. No. 5,917,026. Fusion proteins with CTA1DD any be produced using the vector described in FIG. 7 or as described in references 8, 29.

[0055] The pharmaceutical compositions may comprise one or more immunogenic complexes according to the invention, together with one or more excipients that are acceptable in pharmaceutical or veterinary products, whereby complexes and components to be mixed therewith may be placed in separate compartments.

[0056] The compositions according to the invention will in practice normally be administered orally but may be given topically, or by rectal administration or by injection.

[0057] For oral administration tablets and capsules may contain conventional excipients, such as binders, for example syrup, sorbitol or polyvinyl pyrrolidone; fillers, for example lactose, microcrystalline cellulose, corn starch, calcium phosphate or sorbitol; lubricants, for example magnesium stearate, stearic acid, polyethylene glycol or silica; desintegrants, for example potato starch or sodium starch glycolate, or surfactants, such as sodium lauryl sulphate.

[0058] Oral liquid preparations can be in the form of for example water or oil suspensions, solutions, emulsions, syrups or elixirs, or can be supplied as a dry product for constitution with water or another suitable vehicle before use.

[0059] A composition according to the invention can be formulated for parenteral administration by injection or continuous infusion. Compositions for injection can be provided in unit dose form and can take a form such as suspension, solution or emulsion in oil or aqueous carriers and can contain formulating agents, such as suspending, stabilizing and/or disperging agents. Alternatively, the active constituent can be present in powder form for constitution with a suitable carrier, for example sterile pyrogen-free water, before use.

[0060] The compositions according to the invention can contain between 0.1 and 99% by weight of the active constituent, suitably from 30 to 95% for tablets and capsules and 3 to 50% for liquid preparations.

[0061] The experimental part shows that iscoms containing a fusion protein comprising CTA1-DD linked to the OVA 323-339 peptide epitope, used as a model antigen, were highly immunogenic when given by the subcutaneous, oral or nasal routes, inducing a wide range of systemic T cell dependent immune responses. No toxicity was observed by any route indicating that rationally designed combined vectors consisting of CTA1-DD and iscoms S may provide the basis of potent and safe mucosal vaccines.

[0062] Thus, iscoms containing OVA peptide fused to CTA1-DD were immunogenic when given by a variety of routes, including the oral, nasal and parenteral routes. The responses induced included DTH and serum IgG antibodies in vivo, antigen-specific T cell proliferation and γIFN production in vitro. Despite the fact that it was not possible to detect IL5 production when CTA1-DD-ISCOMS primed lymphocytes were restimulated with OVA in vitro, immunised mice were primed for the production of both IgG2a and IgG1 isotypes, indicating that Th1 and Th2 cells were primed in vivo.

[0063] The immune responses induced by iscoms containing the OVA peptide fused to the intact CTA1-DD construct were markedly superior to those found after immunisation with iscoms containing the CTA1-R72K-DD construct which contains a point mutation that abolishes the enzymatic activity of CTA1. This confirms our previous findings that ADP-ribosylating function is essential for the adjuvant property of the CTA1-DD Vector (8) and indicates that a significant proporon of the combined ISCOMS-CTA1DD structure also depends on targeting this activity to the immune system. Nevertheless, iscoms containing the enzymatically inert CTA1-R72K-DD molecule did retain some adjuvant activity when given by mucosal or parenteral routes. This may reflect the well-established adjuvant properties of the iscoms themselves, perhaps enhanced by the ability of the DD fragment to target them in vivo, presumably to B lymphocytes. Thus, in addition to being targeted to DC and/or macrophages like conventional iscoms (14-16, 17), the new, combined vector may have the additional ability to interact with B cells, creating a second potential source of APC for T cell priming. In addition to its potent APC targeting properties, the interact CTA1-DD-ISCOMS adjuvant has the great advantage of being able to activate these cells, creating a costimulatory microenvironment for efficient T cell priming. Iscoms induce DC and/or macrophages to produce proinflammatory cytokines such as IL1, IL6 and IL12 in vivo (15, 18-22), while CTA1-DD is a potent co-activator of B cells (8). For these reasons, at least three important features of CTA1-DD-ISCOMS were considered to contribute to their immunogenicity. First they can physically target antigen and adjuvant to distinct APC populations in vivo and via distinct mechanisms. In the case of iscoms, this probably involves phagocytic uptake by mononuclear cells, whereas CTA1-DD involves receptor-mediated binding and uptake by surface immunoglobulin (8). Secondly, the vector contains two active adjuvants, Quil A and the ADP-ribosylating enzyme CTA1, which can stimulate the relevant cells that have taken up the vector. Lastly, insertion of the antigenic construct into the rigid iscoms particle ensures that the antigen and the adjuvants ate delivered directly to the same APC, focussing their effects for optimal T cell priming.

[0064] Extremely low doses of OVA peptide were able to prime systemic immunity by both mucosal and parenteral routes using the CTA1-DD-ISCOMS vector, with as little as 150 ng or 750 ng peptide equivalent being effective by the subcutaneous and oral routes respectively. Secondly, although the antigenic epitope used was delivered as part of a large fusion protein inserted in an iscoms particle, it induced strong immune responses that could be recalled with intact OVA protein. This indicates that CTA1-DD fusion protein and the iscoms vector did not interfere with the antigen processing mechanisms, which normally generate this class II MHC-restricted epitope.

[0065] Taken together the results suggest that the combined vector gains access to physiologically relevant antigen processing pathways in an extremely efficient manner. Lastly, it is important to emphasise that no toxicity was observed in mice given the combined adjuvant vectors by any route. This contracts with the toxicity occasionally seen using vectors containing intact Quil A (12, 27, 28), but extends our previous findings that the Quadri A fraction of Quil A and the CTA1-DD fusion protein are themselves lacking significant toxicity, despite their potent adjuvant activities. Thus the combined vector should provide a safe means of inducing mucosal and systemic immunity.

[0066] One surprising finding from the study was at the free CTA1-DD fusion protein also had some adjuvant activity when given by the oral route (FIG. 3). As confirmed here, previous studies had shown that this material was active by parenteral and subcutaneous routes (8), but it was considered it unlikely that it would be able to gain access to the B cells necessary for its adjuvant effects when given into the harsh environment of the intestine. However, it is now shown that oral immunization with CTA1-DD containing a defined peptide epitope induces a wide range of immune responses, which interestingly included marked levels of γIFN, despite other claims that CT based adjuvants stimulate predominantly Th2 dependent responses by this route. The responses induced by free CTA1-OVAp-DD were not as high as those, which occurred, when the fusion protein was inserted in iscoms, underling the added potency of the combined vector. However, the enzymatically inactive CTA-R72K-OVAp protein was unable to induce any response above that generated by peptide alone by the oral or parenteral routes, indicating that the adjuvant properties of the intact CTA1-DD material were dependent on its ADP ribosylating activity, even when give by the oral route.

[0067] Subcutaneous immunisation gives a synergistic between the CTA1-DD and iscom adjuvant effect in proliferation for CTA1-OVAp-DD-ISCOMS over the sum of the proliferation levels of CTA1-OVAp-DD and CTA1-OVAp-R72K-DD (FIG. 2A). Similarly oral immunisation gives a synergistic effect in IgG2a induction for (FIG. 3D) and in proliferation and γIFN induction for (FIGS. 4A and 4B). Also, intranasal administration gives a synergistic effect in proliferation and γIFN induction as can be seen from FIG. 5.

[0068] Together, the results are encouraging evidence that by combining the distinctive adjuvant properties of iscoms and the novel, non-toxic CTA1-DD derivative, it may prove possible to construct effective, safe and stable subunit vaccines which are active by mucosal routes.

[0069] All cited references herein are incorporated by reference. The invention will now be further described by non-limiting specific examples with reference to the appended drawings, wherein:

FIGURE LEGENDS

[0070]FIG. 1:

[0071] Induction of systemic immune responses by subcutaneous immunization with iscoms containing CTA1-OVAp-DD or enzymatically inactive CTA1-R72K-OVAp-DD. Control mice received CTA1-OVAp-DD alone, or OVA 323-339 peptide alone. All mice received th equivalent of 150 ng OVA peptide and results shown are primary DTH responses measured 7 days after immunization (A), serum total IgG (B), IgG1 (C) and IgG2a (D) antibody levels measured 7 days a subcutaneous challenge with soluble OVA given 7 days after primary immunization. The data are means±1 standard deviation for 5 mice/group and are representative of 3 similar experiments (**, p<0.05 vs all other groups; ¶¶, p<0.05 vs OVAp alone).

[0072]FIG. 2:

[0073] Induction of systemic immune responses by subcutaneous immunization with iscoms containing CTA1-OVAp-DD or enzymatically inactive CTA1-R72K-OVAp-DD. Control mice received CTA1-OVAp-DD alone, or OVA 323-339 peptide alone. All mice received the equivalent of 150 ng OVA peptide and results shown are proliferation (A) and γIFN (B) levels measured in draining lymph nodes, measured 7 days after immunization. The data are means±1 standard deviation for 5 mice/group and are representative of 3 similar experiments (**, p<0.05 vs all other groups; ¶¶, p<0.05 vs OVAp alone).

[0074]FIG. 3:

[0075] Induction of systemic immune responses by oral immunization with iscoms containing CTA1-OVAp-DD or enzymatically inactive CTA1-R72K-OVAp-DD. Control mice received CTA1-OVAp-DD alone, CTA1-R72K-OVAp-DD, or OVA 323-339 peptide alone. All mice received the equivalent of 750 ng OVA peptide on 6 occasions and the results shown are primary DTH responses measured 7 days after the last immunization (A), serum total IgG (B), IgG1 (C) and IgG2a (D) antibody levels measured 7 days after a subcutaneous challenge with soluble OVA given 7 days after the last immunization. The data are means±1 standard deviation for 4-5 mice/group and are representative of 3 similar experiments (**, p<0.05 vs all other groups; ¶¶, p<0.05 vs OVAp alone).

[0076]FIG. 4;

[0077] Induction of systemic immune responses by oral immunization with iscoms containing CTA1-OVAp-DD or enzymatically inactive CTA1-R72K-OVAp-DD. Control mice received CTA1-OVAp-DD alone, CTA1-R72K-OVAp-DD, or OVA 323-339 peptide alone. All mice received the equivalent of 750 ng OVA peptide on 6 occasions and the results shown are proliferation (A) and γ IFN (B) levels measured in draining lymph nodes, measured 7 days after immunization. The data are means±1 standard deviation for 45 mice/group and are representative of 3 similar experiments (p*, p<0.05 vs all other groups; ¶¶, p<0.05 vs OVAp alone).

[0078]FIG. 5:

[0079] Priming of systemic T cells by intranasal immunization with iscoms containing CTA1-OVAp-DD or enzymatically inactive CTA1-R7K-OVAp-DD. Control mice received CTA1-OVAp-DD alone or OVA₃₂₃₋₃₃₉ alone. All mice received the equivalent of 150 ng of OVA peptide, and results shown are proliferation (A) and γ IFN (3) levels me ed in the spleen 7 days after immunization. The data are means±1 SD for five mice per group and are representative of tee similar experiments (p*, p<0.05 vs all other groups; ¶¶, p<0.05 vs OVAp alone).

[0080]FIG. 6:

[0081] Construction of iscoms consisting of (A) CTA1-OVAp-DD and PR8, (B) CTA1R7K-OVAp-DD and PR8 and (C) PR8 antigens alone. The CTA1-OVAp-DD and CTA1R17K-OVAp-DD were detected by a HRP (horse radish peroxidase) conjugated rabbit antibody binding to the DD domain and the PR8 antigens were detected using biotinylated chicken anti-PR8 immunoglobulin (not binding to the DD) domain) followed by HRP conjugated streptavidin. The saponins composing the iscom particle were detected by their absorbance at 210 nm.

[0082]FIG. 7:

[0083] Linear map of CTA1-OVA-DD sequence check: 1329 from: 1 to: 3798 CTA1-DD expressions vector with 217 enzymes:*. Max Cuts: 1.

[0084]FIG. 8:

[0085] DNA and amino acid sequence of CTA1-DD fusion protein.

Example 1 Preparation of CTA1DD Fusion Protein

[0086] Preparation of a Cholera Toxin A1 Subunit (CTA1) Fusion Protein was made as described in U.S. Pat. No. 5,917,026.

[0087]Escherichia coli strains HB101 and E. coli RV308 were used as bacterial hosts for all atoning and expression work. Standard plasmids and vectors used were: pUC 19 and the PCR.TM. vector (Invitrogen, USA). Restriction enzymes and T4 DNA ligase (Boehringer Mannheim, Germany and New England Biolabs, USA) were used according to the recommendation of the supplier.

[0088] The oligonucleotides used in the polymerase chain reaction (PCR) were synthesised with an automated machine (Pharmacia-LKB Gene Assembler Plus, Pharmacia Uppsala, Sweden) and phosphorylated separately using polynucleotide kinase (New England Biolabs, USA). Low melting temperate agarose (NUSIEVE.RTM. GTG, FMC Bioproducts, USA) was used to isolate DNA fragments, and Multi Purpose agarose (Boehringer Mannheim, Germany) for DNA analysis.

[0089] The PCR amplifications were performed using the DNA Thermal cycler and Taq DNA polymerase (Perkin-Elmer Cetus Instruments, USA).

[0090] The bacterial strains were grown in Luria Bertani medium (LB) or yeast tryptone medium (2.times.YT) with ampicillin (Ap) 50 .mu.g/ml or kanaycin (Km) 100 .mu.g/ml. Plasmid DNA was prepared according to MAGIC.RTM. Minipreps DNA Purification Systems manual (Promega, USA).

[0091] To determine the nucleotide sequence of the obtained fragments, DNA sequencing was performed using the Sanger method (Sanger, F., Nicklen, S. and Coulson, A. R. DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA. 74, 5463-7, 1977,22). Both strands were sequenced according to a standard protocol for the Taq DYEDEOXY.RTM. Terminator cycle sequencing kit (Applied Biosystems, USA). Analyses were performed on an Applied Biosystems Model 373A DNA Sequencing system.

[0092] The gene encoding cholera toxin A1 subunit amino acids 1 to 186 (Mekalanos, J. J., Swartz, D. J., Pearson, G. D., Harford, N., Groyne, F. and de, W. M. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature. 306, 551-7, 1983) was obtained by PCR using two synthetic DNA primer (1 and 2 in FIG. 1 of U.S. Pat. No. 5,917,026). Similarly the DNA segment encoding the IgG-binding region D of staphylococcus aureus protein A (Uhlen, M., Guss, B., Nilsson B., Gatenbeck, S., Philipson, L. and Lindberg, M. Complete Sequence of the Staphylococcal Gene Encoding Protein A. J. Biol. Chem. 1984) was obtained by PCR using two synthetic DNA primers (3 and 4 in FIG. 1 of U.S. Pat. No. 5,917,026).

[0093] Using standard molecular biology techniques as described in Sambrook, J. and al., e. Molecular Cloning—A Laboratory Manual.; Second edition ed.; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.: 19891, plasmid pKP 1001 (FIG. 2 of U.S. Pat. No. 5,917,026) was constructed. Plasmid pKP 1001 contains the gene encoding CTA1 (aa 1-186) fused in fame with a DNA element encoding two, tandem copies of the D region from S. aureus protein A. In pKP 1001 the transcription unit encoding the CTA1-DD fusion protein is under control of the tryptophane promoter pTrp.

[0094] For the production of the CTA1-DD fusion protein, E. coli RV308 and HB101 cells transformed with plasmid pKP1001 were grown in shaker flasks overnight in 2.times.YT or LB (250 ml or 500 ml), with kanamycin, at 37.degree. C. After culture, the cells were collected by centrifugation. In order to solubilize the intracellularly produced fusion proteins, which precipitated as inclusion bodies, the cell pellet was treated with 6M Guanidine-HCl. After addition of destined water to 1M Guanidine-HCl to allow the protein to refold, the fusion protein was purified by IgG affinity chromatography using IgG Sepharose (Pharmacia, Sweden). After passage of the solubilized fusion protein through the affinity column the gel was washed with TST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20), followed buffered with 10 mM ammonium acetate, pH 4.8. Finally the fusion protein was eluated in 0.2 M acetic acid pH 3.1. The eluted fusion protein was stored in aliquots at −20.degree. C. prior to further use. Two other fusion proteins, one comprising CTA1 fused to DD at its amino terminus (DD-CTA1) and a second (CTA1(Asp109->Ala)-DD) consisting of a mutant form of CTA1 in which Asp 109 was converted to Ala by PCR-directed in vitro mutagenesis, were prepared in the same way.

Example 2 Preparation of Iscoms

[0095] Antigens and Adjuvants

[0096] Ovalbumin (Fraction V) was obtained from Sigma (Poole, UK), while OVA 323-339 peptide was obtained from Sigma Genosys, CTA1-DD and CTA1-R72K-DD fusion proteins containing the OVA 323-339 epitope were prepared as described in references 8 and 29, CTA1-R72K is a variant of CTA1 from which one amino acid has been deleted and which lacks enzymatic activity.

[0097] For the generation of the fusion proteins CTA1-OVAp-DD and CTA1-R7K-OVAp-DD, harbouring one copy of OVA 323-339 between the DD and the CTA1 moieties, a synthetic oligonucleotide encoding OVA 323-339 flanked by nonpalindromic AvaI sites (31) was inserted bead-to-tail into the BbsI site in vectors pCTA1-DD and pCTA1-R7K-DD. For the production of fusion proteins, Escherichia coli TG-1 cells transformed with the different expression vectors were grown in 250 ml flasks overnight in 2×YT or Luria-Bertani, with 50 μg/ml kanamycin, at 37° C. After culture, the cells were collected by centrifugation, and the fusion proteins produced as inclusion bodies, were solubilized by treatment with 6 M guanidine-HCl. After addition of distilled water to allow refolding, the fusion proteins were purified by affinity chromatography on IgG-Sepharose (Pharmacia, Peapack, N.J.) and stored in 0.2 M HAc at 4° C.

[0098] Iscom-CTA1-R7K-OVA-DD and Iscom-CTA1-OVA-DD

[0099] The preparation of these iscoms was done simultaneously. Prom the stock solution of protein (1 mg/ml) in 0.2 M acetic acid pH 4, both recombinant proteins (CTA1-OVA-DD and CTA1-R7K-OVA-DD) were each dialysed in refrigerator (410° C.) against 0.2 M cold phosphate buffer pH 6.

[0100] A one ml sample of each dialysed recombinant solution (equivalent to one mg of each protein) were transferred to room temperature.

[0101] One mg purified freeze-dried saponin fraction (Quadri A), normally kept in sealed glass containers in freezer below −18° C., were also transferred to room temperature opened and the glass container with freeze-dried saponin and a small magnetic bar were arranged with clamps over a magnetic stirrer.

[0102] A stock solution of 1% lipid mix (1% cholesterol and 1% phoshatidylcholine dissolved in 20% Mega 10), normally kept in sealed plastic vials in freezer (below −18° C.), were also transferred to room temperature and melted at hand temperature (30-40° C.).

[0103] The one mL protein sample were added to the freeze-dried saponin and as soon (a few seconds) the saponin were dissolved in the sired protein solution ten 40 microliter of hand warm 1% lipid mix were added and the mixture stirred for the next 3 hours at room temperature. The mixture were then dialysed for the next 2-3 hours at room temperature against 0.2 M room temperate phosphate buffer pH 6. The dialyse was continued in refrigerator (4-10° C.) for one night. Next day the dialysed protein was centrifuged for 5 minutes at 10000×g and the supernatant (0.9 ml) transferred as a 300 microliter overlay to each of three 4 ml ultra centrifugation plastic vials with preformed sucrose freeze gradient. The freeze gradient was on basis of 25% (W/W) sucrose dissolved in 0.2 M phosphate buffer pH 6.

[0104] After 5 hours ultra centrifugation (20° C., sw60 rotor, 50000 rpm, r_(sv)=2570000×g) fractions were collected by puncturing the plastic vial with a needle in the bottom. The fractions were analysed for antigenicity, protein content and density. The protein rich fractions, with no free protein were pooled and dialysed in refrigerator (4-10° C.) for 2 days against 0.2 M phosphate buffer pH 6. Finally the iscom preparation was concentrated using centrifugal filter device until desired protein concentration of 0.5 mg/mL was obtained.

[0105] Iscom-CTA1DD

[0106] This preparation was done as described above using CTA1DD as protein source instead of CTA1-OVA-DD or CTA1-R7K-OVA-DD.

[0107] Iscom-Matrix

[0108] Saponin Fractionation

[0109] Quadri A was prepared as described in Kamstrup S., San Martin R., Doberti A., Grande H., Dalsgaard K: Preparation and characterisation of quillaja saponin with less heterogeneity than Quil-A. Vaccine Vol. 18, No. 21, Apr. 1, 2000.

[0110] From aliquots of 10 mg Quadri A saponin/ml H₂O (kept at −20 C) a sample of 2.5 mg saponin (250 microliter) was added to room temperated 2.183 microliter 0.2 M phosphate buffer pH 6, and placed over magnetic stirrer with small magnet in the same way as described above, Fresh thawn 67 microliter room temperated 1.5% lipidmix (1.5% cholesterol and 1.5% phoshatidylcholine dissolved in 20% Mega 10) were then added.

[0111] After 3-5 hours stirring at room temperature the resulting 2.5 ml product was dialysed at room temperature one night and day against 0.2 M phosphate buffer pH 6. Iscoms containing OVA 323-339 peptide were prepared by EP 97905539.9 and EP 97905541.5. Iscom matrix without protein were prepared by mixing 2.5 mg Quadri A in 250 μl H₂O with 2.183 ml 0.2M PBS pH 6 plus 67 μl lipid mix containing 1.5% cholesterol and 1.5% phoshatdylcholine in 20% Mega10 and sting for 3-5 hours at room temperature. The resulting iscoms were dialysed at room temperature for 36 hours against 0.2 M PBS analysed by EM as above.

Example 3 Subcutan Immunisation

[0112] Animals

[0113] BALB/c mice (H-2d) were purchased from Harlan Olac (Bicester, UK) and maintained under SPF conditions in the Central Research Facility, University of Glasgow, or were obtained from B&K (Sollentuna, Sweden) and bred in the Department of Microbiology and Immunology, University of Gothenburg. All animals were first used at 6-8 weeks of age.

[0114] Immunisaton of Animals

[0115] Mice were immunised subcutaneously into one footpad with iscoms or purified fusion proteins containing 4 μg of CTA1-OVAp-DD, equivalent to 150 ng OVA 323-339 in a total volume of 50 μl. One group of mice received 150 ng OVA 323-339 alone.

[0116] Measurement of OVA-Specific Immune Responses In Vivo

[0117] 7 days after the last immunisation, delayed type hypersensitivity (DTH) were assessed by detecting the increment in footpad thickness found 24 hours after subcutaneous injection of 100 μg of heat aggregated OVA in 50 μl of sterile saline. Mice were bled for primary serum antibody responses at this time and also 7 days after DTH challenge to assess secondary responses. Secretory IgA antibody responses were measured 7-10 days after the last feed of antigen in intestinal washes obtained after four gavages with PEG as described previously (9). Total IgG, IgA and IgG1 and IgG2a isotype responses were measured by ELISA, as described previously (10). Local antibody responses were measured in the lung.

[0118] Measurement of OVA-Specific Immune Responses In Vitro

[0119] 7 days after the last immunisation, draining popliteal lymph nodes, spleens or cervical lymph nodes were removed and single cell suspensions prepared in RPMI 1640 (Gibco BRL, Paisley, Scotland) by rubbing through a stainless steel mesh and passing the resulting suspension through Nitex mesh (Cadisch & Sons, London, U.K.). Alter tree washes in medium, the cells were resuspended at a final concentration of 10⁶ cells/ml ad cultured in 200 μl aliquots in flat bottomed 96 well tissue culture plates (Costar, Nucleopore, High Wycombe, U.K.) in RPMI 1640 containing 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml fungizone, 2 mM L-glutamine, 25 mM Hepes, 50 mM 2-mercaptoethanol (all Gibco BRL), either alone or with 1 mg/ml OVA. Proliferation was assessed by addition of 1 μCi/well ³H thymidine for the last 18 hours of culture. Cell bound DNA was harvested on filter mats and ³H-TdR incorporation measured on a Betaplate counter. To measure cytokine production, 4×10⁶ lymph node cells in 1 ml aliquots were cultured in 24 well tissue culture plates (Costar) either in medium alone or with 1 mg/ml OVA, Supernatants were harvested after 24 days and stored at −20° C. until assayed. Cytokine production was quantified using sandwich ELISA techniques described in detail elsewhere (3, 21), using appropriate pairs of capture and biotinylated detection antibodies (all Pharmingen). Antibody binding was detected using extravidin-peroxidase (Sigma) and TMB substrate as described above. Cytokine concentrations in test supernatants were determined with reference to a standard curve constructed using serial dilutions of recombinant cytokines (Pharmingen).

[0120] Statistical Analysis

[0121] Results are expressed as means+/− 1 SD and comparisons were made using unpaired two tailed Student's t-test.

[0122] Results

[0123] Incorporation into Iscoms Enhances the Systemic Immunogenicity of CTA1-DD Adjuvant Vector

[0124] A. In Vivo Responses

[0125] Having successfully incorporated the CTA1-OVAp-DD fusion proteins into iscoms, the immunogenicity of the combined vector was compared with the intact fusion protein. Mice were immunised subcutaneously on one occasion and the subsequent systemic immune responses assessed by measuring primary OVA-specific DTH responses, primary and secondary serum antibody responses and in vitro T cell responses in the draining lymph node.

[0126] As anticipated, mice immunised with 150 ng OVA 323-339 in saline showed little or no DTH response above background, whereas mice immunised with purified CTA1-OVAp-DD fusion protein containing the same amount of OVA 323-339 had good DTH responses (FIG. 1A). However, animals receiving CTA1-OVAp-DD fusion protein incorporated it ISCOMS had significantly enhanced DTH responses compared with CTA1-OVAp-DD immunised mice (FIG. 1A). Confirming our previous findings that the enzymatically inactive CTA1-R72K fusion protein lacks inherent adjuvant activity (8), mice receiving CTA1-R72K-OVAp alone had no significant DTH responses above background. However, iscoms containing this material were immunogenic, inducing significant primary DTH responses that were markedly less than those found in mice immunised with CTA1-OVAp-DD-ISCOMS (FIG. 1A), presumably reflecting the known adjuvant properties of the iscom vector itself.

[0127] Primary serum antibodies reactive with native OVA could not be detected after immunisation with OVAp in any form (data not shown). However, secondary total IgG antibody responses did occur after subcutaneous challenge with heat aggregated OVA in animals primed with immunogenic vectors. These followed a similar pa to the DTH responses, with the highest levels of IgG anti-OVA being found in mice immunised with CTA1-OVAp-DD-ISCOMS (FIG. 1B). Significant but lower, IgG responses also occurred in mice given intact CTA1-OVAp-DD fusion protein and in animals receiving CTA1-OVAp-R72K-DD-ISCOMS, whereas mice immunised with OVAp alone or enzymatically inactive CTA1-OVAp-R72K-DD had little or no total IgG antibody in serum (FIG. 1B).

[0128] Interestingly, immunisation with CTA1-OVAp-DD-ISCOMS or with CTA1-OVAp-DD primed for both IG1 and IgG2a antibody responses (FIGS. 1C/D), suggesting no bias towards priming of Th1 or Th2 cells by the vectors. Again, some IgG1 and IgG2a responses were found in mice given CTA1-OVAp-R72K-DD-ISCOMS, but not in mice immunised with CTA1-OVAp-R72K-DD itself or with OVAp alone.

[0129] B. T Cell Responses In Vitro

[0130] Next the immunogenicity of the combined adjuvant vectors was explored in more detail by examining their ability to primed T cell proliferation and cytokine production. Draining popliteal lymph nodes were removed 7 days after primary immunisation and lymphocytes restimulated in vitro with native OVA.

[0131] As before, mice immunised with CTA1-OVAp-DD-ISCOMS were primed for very strong T cell proliferation FIG. 2A) and production of γ IFN (FIG. 2B). Little or no IL5 production was observed. Immunisation with CTA1-OVAp-DD alone, or with iscoms containing the enzymatically inactive CTA1-R72K fusion protein also induced good T cell responses in vitro, although these were significantly lower than those found in CTA1-OVAp-DD-ISCOMS primed animals. Little or no proliferation or cytokine production was observed in mice receiving OVAp alone (FIG. 2). There is a synergistic effect in proliferation for CTA1-OVAp-DD-ISCOMS over the sum of the proliferation levels of CTA1-OVAp-DD and CTA1-OVAp-R72 K-DD (FIG. 2A)

[0132] Together, these results confirm that enzymatically active CTA1-DD is an effective adjuvant for a broad range of systemic immune responses when given by parenteral routes and extend earlier findings by showing that incorporation into iscoms markedly enhances this activity.

Example 4 Oral Immunisation

[0133] Animals were the same as used in example 3. For oral immunisation, mice were fed on days 1, 2, 3, 8, 9 & 10 with iscoms or-purified fusion proteins containing 41 g of CTA1-OVAp-DD, equivalent to 150 ng OVA 323-339. One group of mice received 750 ng OVA 323-339 on each occasion. In vivo and in vivo measurements were performed as in example 3

[0134] Results

[0135] Incorporation into Iscoms Enhances the Mucosal Immunogenicity of CTA1-DD Adjuvant Vector

[0136] Earlier work show that CTA1-DD functions poorly as an adjuvant by the oral route. However, iscoms are extremely effective when given orally and therefore it was examined if incorporation into iscoms could improve the mucosal adjuvant properties of CTA1-DD.

[0137] A In Vivo Responses

[0138] Mice were fed on six occasions with the different vectors, a protocol found to be optimal in previous work with iscoms, and systemic immune responses assessed as described above. Oral immunisation with CTA1-OVAp-DD-ISCOMS induced significant DTH responses compared with background levels and these were equivalent to those obtained subcutaneous priming (FIG. 3A). In these experiments, CTA1-DD itself also primed systemic DTH by the oral route, although to a significantly lesser degree than when incorporated into iscoms. As with the parenteral route, enzymatically inactive CTA1-R72K fusion protein lacks inherent adjuvant activity and mice receiving CTA1R72K-OVAp orally induced no significant DTH responses, but this material was immunogenic when incorporated into iscoms, confirming the inherent mucosal adjuvant properties of iscoms. Again this response was markedly less than that found with iscoms containing intact CTA1-DD protein (FIG. 3A). Mice fed 150 ng OVA 323-339 in saline showed little or no DTH response above background, whereas mice immunised with purified CTA1-OVAp-DD fusion protein containing the same amount of OVA 323-339 had good DTH responses. However, animals receiving CTA1-OVAp-DD fusion protein incorporated in iscoms had significantly enhanced DTH responses compared with CTA1-OVAp-DD immunised mice (FIG. 3A).

[0139] Primary serum antibodies reactive with native OVA could not be detected after immunisation with OVAp in any form (data not shown). However, secondary total IgG antibody responses did occur after subcutaneous challenge with heat aggregated OVA in animals primed with immunogenic vectors. These followed a similar pattern to the DTH responses, with the highest levels of Ig anti-OVA being found in mice immunised with CTA1-OVAp-DD-ISCOMS (FIG. 3B). Significant, but lower, IgG responses also occurred in mice given intact CTA1-OVAp-DD fusion protein and in animals receiving CTA1-OVAp-R72K-DD-ISCOMS, whereas mice immunised with OVAp alone or enzymatically inactive CTA1-OVAp-R72K-DD had little or no total IgG antibody in serum (FIG. 3B).

[0140] Interestingly, immunisation with CTA1-OVAp-DD-ISCOMS or with CTA1-OVAp-DD primed for both IgG1 and IgG2a antibody responses (FIGS. 3C & D), suggesting no bias towards priming of Th1 or Th2 cells by the vectors. Again, some IgG1 and IgG2a responses were found in mice given CTA1-OVAp-R72K-DD-ISCOMS, but not in mice immunised with CTA1-OVAp-R72K-DD itself or with OVAp alone. There is a synergistic effect in IgG2a induction for CTA1-OVAp-DD-ISCOMS over the sum of the IgG2a levels of CTA1-OVAp-DD and CTA1-OVAp-R72K-DD (FIG. 3D)

[0141] B, T Cell Responses In Vitro

[0142] As before, mice immunised with CTA1-OVAp-DD-ISCOMS were primed for very tong T cell proliferation and production of γIFN (FIGS. 4A & 4B). Immunisation with CTA1-OVAp-DD alone, or with iscoms containing the enzymatically inactive CTA1-R72K fusion protein also induced good T cell responses in vitro, although these were significantly lower than those found in CTA1-OVAp-DD-ISCOMS primed animals. Little or no proliferation or cytokine production was observed in mice receiving OVAp alone. There are synergistic effects in proliferation and γIFN induction for CTA1-OVAp-DD-ISCOMS over the sum of the proliferation and γIFN levels respectively of CTA1-OVAp-DD and CTA1-OVAp-R72K-DD (FIGS. 4A and 4B)

[0143] The results show that a targeted CT derivative can be incorporated into iscoms. The resulting combined vector is a potent adjuvant for inducing a wide range of immune responses to small amounts of peptide immunogen after mucosal and parenteral administration.

Example 5 Intranasal Immunisation

[0144] Mice were immunized intranasally on three occasions 10 days apart, with iscoms or purified fusion proteins containing 4 μg of CTA1-OVAp-DD or CTA1R7K-OVAp-DD (equivalent to 150 ng of OVA₃₂₃₋₃₃₉) in a total volume of 20 μl. Control groups of mice All mice received the equivalent of 150 ng of OVA peptide. FIG. 5 shows the proliferation (A) and IFN-γ (B) levels measured in the spleen 7 days after immunization. It can be seen that there is a synergistic effect in that the level of proliferation and production of IFN-γ when CTA1-OVAp-DD iscoms are used is higher than the sum of the corresponding levels when CTA1R7K-OVAp-DD iscoms are used and CTA1-OVAp-DD.

Exampel 6 Mixed Iscoms Containing Two Antigens

[0145] Mixed iscoms containing both CTA1-OVAp-DD and additional antigens (haemagglutinin and neuraminidase from human influenza virus) were prepared essentially according to Example 2.

[0146] (A) 0.5 mg of each CTA1R7K-OVAp-DD and detergent solubilized PR8 antigens were mixed with 5 mg of saponin (Spikoside, Isconova, Sweden; 100 mg/ml in distilled water) and 1 mg of each cholesterol and phosphatidyl choline (15 mg/ml in 20% MEGA-10).

[0147] (B) 0.5 mg of each CTA1-OVAp-DD and detergent solubilized PR8 antigens were mixed with 5 mg of saponin (Spikoside, Isconova, Sweden; 100 mg/ml in distilled water) and 1 mg of each cholesterol and phosphatidyl choline (15 mg/ml in 20% MEGA-10).

[0148] (C) 1.0 mg of detergent solubilized PR8 antigens were mixed with 5 mg of san (Spikoside, Isconova, Sweden; 100 mg/ml in distilled water) and 1 mg of each cholesterol and phosphatidyl choline (15 mg/ml in 20% MEGA-10).

[0149] The Iscoms were prepared as known in the art (ref. 34). Aliquots of both preparations were analysed by sucrose density gradient centrifugation. The gradients were divided into fractions that were analysed for saponin content (A210) and the two antigens using ELISA. As shown in FIG. 6 both antigens were simultaneously incorporated into the same complex.

[0150] Mice ware immunised subcutaneously and orally as described in Exampel 2, Antibody and systemic T cell responses were recorded against OVA and PR8. As shown for OVA in example 2, the response (antibody IgG1 and IgG2a, proliferation and IFN-γ production were enhanced significantly by the simultaneous incorporation of CTA1-OVAp-DD molecule compared to iscoms containing PR8 antigens alone or combined with CTA1R7K-OVAp-DD.

REFERENCES

[0151] 1, Mowat, A. M. 1999. Oral tolerance: basic mechanisms and clinical applications. Curr Opinion Gastroenterol 15:546.

[0152] 2. Strobel, S., and A. M. Mowat. 1098. Immune responses to dietary antigens: Oral tolerance. Immunology Today 19:173.

[0153] 3. Grdic, D., R. E. Smith, A. M. Donachie, M. Kjerrulf, E. Hornquist, A. M. Mowat, and N. Lycke. 1999. The mucosal adjuvant effect of cholera toxin and ISCOMS differ in their requirement for IL-12, indicating different pathways of action. Eur J Immunol 29:1774.

[0154] 4. Wilson, A. D., M. Bailey, N. A. Williams and C. R. Stokes. 1991. The in vitro production of cytokines by mucosal lymphocytes immunized by oral administration of keyhole limpet hemocyanin using cholera toxin as an adjuvant. Eur. J. Immunol 21:2333.

[0155] 5. McGhee, J. R., C. Czerkinsky, and J. Mestecky. 1999. Mucosal vaccines: an overview. In Mucosal immunology, 2nd edition. P. L. Ogra, 3J Mestecky, M. B. Lamm W. Strober, J. R. McGhee, and J. Bienenstock, eds. Academic Press, San Diego, p. 741.

[0156] 6, McGhee, J. R., J. Mestecky, M. T. Dertzbaugh, J. H. Eldridge, M. Hirasawa, and H. Kiyono. 1992. The mucosal immune system: from fundamental concepts to vaccine development, Vaccine 10:75.

[0157] 7. Elson, C. O., and M. T. Dertzbaugh. 1999. Mucosal Adjuvants. In Mucosal Immunology 2nd Edition. P. L. Ogra, I. Mestecky, M. E. Lamm, W. Strober, J. R, McGhee, and J. Bienenstock, eds. Academic Press, San Diego, p. 818.

[0158] 8. Agren, L. C., L. Ekman, B. Lowenadler, J. G. Nedrud, and N. Lycke. 1999, Adjuvanticity of the cholera toxin A1-based gone fusion protein, CTA1-DD, is critically dependent on the ADP-ribosyltransferase and Ig-binding activity. J. Immunol 162:2432.

[0159] 9. Mowat, A. M., K. J. Maloy, and A. M. Donachie. 1993. Immune stimulating complexes as adjuvants for inducing local and sync immunity after oral immunisation with protein antigens. Immunology 80:527.

[0160] 10. Maloy, K. J., A. M. Donachie, and A. M. Mowat. 1995. Induction of Th1 and Th2 CD4⁺ T cell responses by oral or parenteral immunization with ISCOMS. Eur J Immunol 2:2835.

[0161] 11. Smith, R. B., A. M. Donachie, and A. M. Mowat. 1998. Immune stimulating complexes as mucosal adjuvants. Immunol Cell Biol 76:263.

[0162] 12. Sjolander, A., J. C. Cox, and I. G. Barr. 1998. ISCOMs: an adjuvant with multiple functions. J Leuk Biol 64:713.

[0163] 13, Vajdy, M., M. H; Kosco-Vilbois, M. Kopf, G. Kohler, and N. Lycke. 1995. Impaired mucosal immune responses in Interleukin 4-targeted mice. J. Exp. Med. 181:41.

[0164] 14. Claassen, I. J. T. M., A. D. M. E. Osterhaus, and E. Claassen. 1995. Antigen detection in vivo after on with different presentation forms of rabies antigen: Involvement of marginal metallophilic metaophages in the uptake of immune-stimulating complexes. Eur J Immunol 25:1446.

[0165] 15. Claassen, I. J. T. M., A. D. M. E. Osterhaus, M. Poelen, N. Van Rooijen, and E. Claassen, 1998. Antigen detection in vivo after immunization with different presentation forms of rabies virus antigen. II. Cellular, but not humoral systemic immune responses against rabies virus immune stimulating complexes are macrophage dependent Immunology 94:455.

[0166] 16. Watson, D. L., N. A Watson, C. Fossum, K. Lovgren, and B. Morein. 1992. Interactions between immune-stimulating complexes (ISCOMs) and peritaneal mononuclear leucocytes. Microbiol Immunol 36:199.

[0167] 17. Smith, R. E., A. M. Donachie, D. Grdic, N. Lycke, and A. M. Mowat. 1999. Induction of innate immune responses by immune simulating complexes: a critical role for IL12 in the immunogenicity of ISCOMS. J Immunology 162:5536.

[0168] 18. Smith, R. E., A. M. Donachie, F. H. McLaren, and A. M. Mowat. 1998. Preservation of mucosal and systemic adjuvant properties of ISCOMS in the absence of functional interleukin 4 or g interferon. Immunology 93:556.

[0169] 19. Villacres-Eriksson, M., S. Behboudi, A. J. Morgan, G. Trinchieri, and B. Morein. 1997. Immunomodulation by Quillaja saponaria ad{acute over (j)}uvant formulations: in vivo stimulation of interleukin 12 and its effects on the antibody response. Cytokine 9:73.

[0170] 20. Behboudi, S., B. Morein, and M. Villas-Eriksson. 1997. In vivo and in vitro induction of IL-6 by Quillaja saponaria molina triterpenoid formulations. Cytokine 9:682.

[0171] 21. Behboudi, S., B. Morein, and M. Villacres-Eriksson. 1996. In vitro activation of antigen-presenting cells (APC) by defined composition of Quillaja saponaria Molina triterpenoids. Clinical & Experimental Immunology 105:26.

[0172] 22. Mowat, A. M., R. E. Smith, A. M. Donachie, E. Furrie, D. Grdic, and N. Lycke. 1999. Oral vaccination with Immune stimulating complexes. Immunology Letters 65:133.

[0173] 23. Kamstrup, S., R, San Martin, A. Doberti, H. Grande, and K. Dalsgaard 2000. Preparation and characterisation of Quillaja saponin with less heterogeneity than Quil-A. Vaccine 18.

[0174] 24, Heeg, K., W. Kuon, and H. Wagner. 1991. Vaccination of Class I major histocompatability complex (MHC)-restricted murine CD8+ cytotoxic T lymphocytes toward soluble antigen: immunostimulating complexes enter the Class I MHC-restricted antigen pathway and allow sensitization against the immunodominant peptide. Eur. J. Immunol, 21:1523.

[0175] 25. Takahashi, H., T. Takeshita, B. Morein, S. Putney, R. N. Germain, and J. Berzofsky. 1990. Induction of CD8⁺ cytotoxic T cells by immunisation with purified HIV-1 envelope protein in ISCOMS. Nature 344:873.

[0176] 26. Mowat, A. M., A. M. Donachie, G. Reid, and O. Jarrett. 1991. Immune stimulating complexes containing Quil A and protein antigen prime Class I MHC-restricted T lymphocytes in vivo and are active by the oral route. Immunology 72:317.

[0177] 27. Morein, B., K. Lovgren, B. Ronnberg, A. Sjolander, and M. Villacres-Erkisson 1995. Immunostimulating complexes. Clinical potential in vaccine development. Clin. Immunother. 3:461.

[0178] 28. Johansson, M., K. Morein and K. Lovgren-Bengtsson. 1999. Iscoms with different Quillaja saponin components differ in their immunomodulating activities. Cell Immunol: In Press.

[0179] 29. Agren, L. C, Ekman, B. Lowenadler and N. Y. Lycke 1997. Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit. J. Immunol. 158:3936.

[0180] 30. Lovgren, K., Kaberg, H. and Morein, B. (1990). An experimental influenza subunit vaccine (ISCOM)—induction of protective immunity to challenge infection in mice after intranasal or subcutaneous administration Clin. exp. Immunol. 82, 435-439.

[0181] 31. Lowenadler, B., A. M. Svennerholm, M. Gidlund, B. Holmgren, K. Krook, C. Svanholm S. Ulf. and S. Josephson, 1990. Enhanced immunogenicity of recombinant peptide fusions comprising multiple copies of a heterologous T helper epitope. Euro. J. Immunol, 20:1541.

[0182]

1 6 1 1029 DNA Artificial Sequence Description of Artificial Sequence DNA encoding CTA1-DD fusion protein 1 atg aaa gca att ttc gta ctg aaa gct tct aat gat gat aag tta tat 48 Met Lys Ala Ile Phe Val Leu Lys Ala Ser Asn Asp Asp Lys Leu Tyr 1 5 10 15 cgg gca gat tct aga cct cct gat gaa ata aag cag tca ggt ggt ctt 96 Arg Ala Asp Ser Arg Pro Pro Asp Glu Ile Lys Gln Ser Gly Gly Leu 20 25 30 atg cca aga gga cag agt gag tac ttt gac cga ggt act caa atg aat 144 Met Pro Arg Gly Gln Ser Glu Tyr Phe Asp Arg Gly Thr Gln Met Asn 35 40 45 atc aac ctt tat gat cat gca aga gga act cag acg gga ttt gtt agg 192 Ile Asn Leu Tyr Asp His Ala Arg Gly Thr Gln Thr Gly Phe Val Arg 50 55 60 cac gat gat gga tat gtt tcc acc tca att agt ttg aga agt gcc cac 240 His Asp Asp Gly Tyr Val Ser Thr Ser Ile Ser Leu Arg Ser Ala His 65 70 75 80 tta gtg ggt caa act ata ttg tct ggt cat tct act tat tat ata tat 288 Leu Val Gly Gln Thr Ile Leu Ser Gly His Ser Thr Tyr Tyr Ile Tyr 85 90 95 gtt ata gcc act gca ccc aac atg ttt aac gtt aat gat gta tta ggg 336 Val Ile Ala Thr Ala Pro Asn Met Phe Asn Val Asn Asp Val Leu Gly 100 105 110 gca tac agt cct cat cca gat gaa caa gaa gtt tct gct tta ggt ggg 384 Ala Tyr Ser Pro His Pro Asp Glu Gln Glu Val Ser Ala Leu Gly Gly 115 120 125 att cca tac tcc caa ata tat gga tgg tat cga gtt cat ttt ggg gtg 432 Ile Pro Tyr Ser Gln Ile Tyr Gly Trp Tyr Arg Val His Phe Gly Val 130 135 140 ctt gat gaa caa tta cat cgt aat agg ggc tac aga gat aga tat tac 480 Leu Asp Glu Gln Leu His Arg Asn Arg Gly Tyr Arg Asp Arg Tyr Tyr 145 150 155 160 agt aac tta gat att gct cca gca gca gat ggt tat gga ttg gca ggt 528 Ser Asn Leu Asp Ile Ala Pro Ala Ala Asp Gly Tyr Gly Leu Ala Gly 165 170 175 ttc cct ccg gag cat aga gct tgg agg gaa gag ccg tgg att cat cat 576 Phe Pro Pro Glu His Arg Ala Trp Arg Glu Glu Pro Trp Ile His His 180 185 190 gca ccg ccg ggt tgt ggg aat gct cca aga tca tcg gga tcc ggg aag 624 Ala Pro Pro Gly Cys Gly Asn Ala Pro Arg Ser Ser Gly Ser Gly Lys 195 200 205 aca ccc gag gct gat gcg caa caa aat aac ttc aac aaa gat caa caa 672 Thr Pro Glu Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln 210 215 220 agc gcc ttc tat gaa atc ttg aac atg cct aac tta aac gaa gcg caa 720 Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln 225 230 235 240 cgt aac ggc ttc att caa agt ctt aaa gac gac cca agc caa agc act 768 Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr 245 250 255 aac gtt tta ggt gaa gct aaa aaa tta aac gaa tct caa gca ccc aaa 816 Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys 260 265 270 ccc gag gct gat gcg caa caa aat aac ttc aac aaa gat caa caa agc 864 Pro Glu Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln Ser 275 280 285 gcc ttc tat gaa atc ttg aac atg cct aac tta aac gaa gcg caa cgt 912 Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg 290 295 300 aac ggc ttc att caa agt ctt aaa gac gac cca agc caa agc act aac 960 Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn 305 310 315 320 gtt tta ggt gaa gct aaa aaa tta aac gaa tct caa gca ccc aaa ccc 1008 Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Pro 325 330 335 gag gta gca ggt cag aat tag 1029 Glu Val Ala Gly Gln Asn 340 2 342 PRT Artificial Sequence Description of Artificial Sequence CTA1-DD fusion protein 2 Met Lys Ala Ile Phe Val Leu Lys Ala Ser Asn Asp Asp Lys Leu Tyr 1 5 10 15 Arg Ala Asp Ser Arg Pro Pro Asp Glu Ile Lys Gln Ser Gly Gly Leu 20 25 30 Met Pro Arg Gly Gln Ser Glu Tyr Phe Asp Arg Gly Thr Gln Met Asn 35 40 45 Ile Asn Leu Tyr Asp His Ala Arg Gly Thr Gln Thr Gly Phe Val Arg 50 55 60 His Asp Asp Gly Tyr Val Ser Thr Ser Ile Ser Leu Arg Ser Ala His 65 70 75 80 Leu Val Gly Gln Thr Ile Leu Ser Gly His Ser Thr Tyr Tyr Ile Tyr 85 90 95 Val Ile Ala Thr Ala Pro Asn Met Phe Asn Val Asn Asp Val Leu Gly 100 105 110 Ala Tyr Ser Pro His Pro Asp Glu Gln Glu Val Ser Ala Leu Gly Gly 115 120 125 Ile Pro Tyr Ser Gln Ile Tyr Gly Trp Tyr Arg Val His Phe Gly Val 130 135 140 Leu Asp Glu Gln Leu His Arg Asn Arg Gly Tyr Arg Asp Arg Tyr Tyr 145 150 155 160 Ser Asn Leu Asp Ile Ala Pro Ala Ala Asp Gly Tyr Gly Leu Ala Gly 165 170 175 Phe Pro Pro Glu His Arg Ala Trp Arg Glu Glu Pro Trp Ile His His 180 185 190 Ala Pro Pro Gly Cys Gly Asn Ala Pro Arg Ser Ser Gly Ser Gly Lys 195 200 205 Thr Pro Glu Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln 210 215 220 Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln 225 230 235 240 Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr 245 250 255 Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys 260 265 270 Pro Glu Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln Ser 275 280 285 Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg 290 295 300 Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn 305 310 315 320 Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Pro 325 330 335 Glu Val Ala Gly Gln Asn 340 3 3798 DNA Artificial Sequence Description of Artificial Sequence Synthetic expression vector 3 gtg cct cac tga tta agc att ggt aac tgt cag acc aag ttt act cat 48 Val Pro His Leu Ser Ile Gly Asn Cys Gln Thr Lys Phe Thr His 1 5 10 15 ata tac ttt aga ttg att taa aac ttc att ttt aat tta aaa gga tct 96 Ile Tyr Phe Arg Leu Ile Asn Phe Ile Phe Asn Leu Lys Gly Ser 20 25 30 agg tga aga tcc ttt ttg ata atc tca tga cca aaa tcc ctt aac gtg 144 Arg Arg Ser Phe Leu Ile Ile Ser Pro Lys Ser Leu Asn Val 35 40 agt ttt cgt tcc act gag cgt cag acc ccg tag aaa aga tca aag gat 192 Ser Phe Arg Ser Thr Glu Arg Gln Thr Pro Lys Arg Ser Lys Asp 45 50 55 ctt ctt gag atc ctt ttt ttc tgc gcg taa tct gct gct tgc aaa caa 240 Leu Leu Glu Ile Leu Phe Phe Cys Ala Ser Ala Ala Cys Lys Gln 60 65 70 aaa aac cac cgc tac cag cgg tgg ttt gtt tgc cgg atc aag agc tac 288 Lys Asn His Arg Tyr Gln Arg Trp Phe Val Cys Arg Ile Lys Ser Tyr 75 80 85 90 caa ctc ttt ttc cga agg taa ctg gct tca gca gag cgc aga tac caa 336 Gln Leu Phe Phe Arg Arg Leu Ala Ser Ala Glu Arg Arg Tyr Gln 95 100 105 ata ctg tcc ttc tag tgt agc cgt agt tag gcc acc act tca aga act 384 Ile Leu Ser Phe Cys Ser Arg Ser Ala Thr Thr Ser Arg Thr 110 115 ctg tag cac cgc cta cat acc tcg ctc tgc taa tcc tgt tac cag tgg 432 Leu His Arg Leu His Thr Ser Leu Cys Ser Cys Tyr Gln Trp 120 125 130 ctg ctg cca gtg gcg ata agt cgt gtc tta ccg ggt tgg act caa gac 480 Leu Leu Pro Val Ala Ile Ser Arg Val Leu Pro Gly Trp Thr Gln Asp 135 140 145 gat agt tac cgg ata agg cgc agc ggt cgg gct gaa cgg ggg gtt cgt 528 Asp Ser Tyr Arg Ile Arg Arg Ser Gly Arg Ala Glu Arg Gly Val Arg 150 155 160 165 gca cac agc cca gct tgg agc gaa cga cct aca ccg aac tga gat acc 576 Ala His Ser Pro Ala Trp Ser Glu Arg Pro Thr Pro Asn Asp Thr 170 175 180 tac agc gtg agc tat gag aaa gcg cca cgc ttc ccg aag gga gaa agg 624 Tyr Ser Val Ser Tyr Glu Lys Ala Pro Arg Phe Pro Lys Gly Glu Arg 185 190 195 cgg aca ggt atc cgg taa gcg gca ggg tcg gaa cag gag agc gca cga 672 Arg Thr Gly Ile Arg Ala Ala Gly Ser Glu Gln Glu Ser Ala Arg 200 205 210 ggg agc ttc cag ggg gaa acg cct ggt atc ttt ata gtc ctg tcg ggt 720 Gly Ser Phe Gln Gly Glu Thr Pro Gly Ile Phe Ile Val Leu Ser Gly 215 220 225 ttc gcc acc tct gac ttg agc gtc gat ttt tgt gat gct cgt cag ggg 768 Phe Ala Thr Ser Asp Leu Ser Val Asp Phe Cys Asp Ala Arg Gln Gly 230 235 240 ggc gga gcc tat gga aaa acg cca gca acg cgg cct ttt tac ggt tcc 816 Gly Gly Ala Tyr Gly Lys Thr Pro Ala Thr Arg Pro Phe Tyr Gly Ser 245 250 255 tgg cct ttt gct ggc ctt ttg ctc aca tgt tct ttc ctg cgt tat ccc 864 Trp Pro Phe Ala Gly Leu Leu Leu Thr Cys Ser Phe Leu Arg Tyr Pro 260 265 270 275 ctg att ctg tgg ata acc gta tta ccg cct ttg agt gag ctg ata ccg 912 Leu Ile Leu Trp Ile Thr Val Leu Pro Pro Leu Ser Glu Leu Ile Pro 280 285 290 ctc gcc gca gcc gaa cga ccg agc gca gcg agt cag tga gcg agg aag 960 Leu Ala Ala Ala Glu Arg Pro Ser Ala Ala Ser Gln Ala Arg Lys 295 300 305 cgg aag agc gcc caa tac gca aac cgc ctc tcc ccg cgc gtt ggc cga 1008 Arg Lys Ser Ala Gln Tyr Ala Asn Arg Leu Ser Pro Arg Val Gly Arg 310 315 320 ttc att aat gca gag cgg ccg cct caa ggc gca ctc ccg ttc tgg ata 1056 Phe Ile Asn Ala Glu Arg Pro Pro Gln Gly Ala Leu Pro Phe Trp Ile 325 330 335 atg ttt ttt gcg ccg aca tca taa cgg ttc tgg caa ata ttc tga aat 1104 Met Phe Phe Ala Pro Thr Ser Arg Phe Trp Gln Ile Phe Asn 340 345 350 gag ctg ttg aca att aat cat cga act agt taa cta gta cgc aag ttc 1152 Glu Leu Leu Thr Ile Asn His Arg Thr Ser Leu Val Arg Lys Phe 355 360 365 acg taa aaa ggg tat cga caa tga aag caa ttt tcg tac tga aag ctt 1200 Thr Lys Gly Tyr Arg Gln Lys Gln Phe Ser Tyr Lys Leu 370 375 380 cta atg atg ata agt tat atc ggg cag att cta gac ctc ctg atg aaa 1248 Leu Met Met Ile Ser Tyr Ile Gly Gln Ile Leu Asp Leu Leu Met Lys 385 390 395 taa agc agt cag gtg gtc tta tgc caa gag gac aga gtg agt act ttg 1296 Ser Ser Gln Val Val Leu Cys Gln Glu Asp Arg Val Ser Thr Leu 400 405 410 acc gag gta ctc aaa tga ata tca acc ttt atg atc atg caa gag gaa 1344 Thr Glu Val Leu Lys Ile Ser Thr Phe Met Ile Met Gln Glu Glu 415 420 425 ctc aga cgg gat ttg tta ggc acg atg atg gat atg ttt cca cct caa 1392 Leu Arg Arg Asp Leu Leu Gly Thr Met Met Asp Met Phe Pro Pro Gln 430 435 440 tta gtt tga gaa gtg ccc act tag tgg gtc aaa cta tat tgt ctg gtc 1440 Leu Val Glu Val Pro Thr Trp Val Lys Leu Tyr Cys Leu Val 445 450 455 att cta ctt att ata tat atg tta tag cca ctg cac cca aca tgt tta 1488 Ile Leu Leu Ile Ile Tyr Met Leu Pro Leu His Pro Thr Cys Leu 460 465 470 acg tta atg atg tat tag ggg cat aca gtc ctc atc cag atg aac aag 1536 Thr Leu Met Met Tyr Gly His Thr Val Leu Ile Gln Met Asn Lys 475 480 485 aag ttt ctg ctt tag gtg gga ttc cat act ccc aaa tat atg gat ggt 1584 Lys Phe Leu Leu Val Gly Phe His Thr Pro Lys Tyr Met Asp Gly 490 495 500 atc gag ttc att ttg ggg tgc ttg atg aac aat tac atc gta ata ggg 1632 Ile Glu Phe Ile Leu Gly Cys Leu Met Asn Asn Tyr Ile Val Ile Gly 505 510 515 gct aca gag ata gat att aca gta act tag ata ttg ctc cag cag cag 1680 Ala Thr Glu Ile Asp Ile Thr Val Thr Ile Leu Leu Gln Gln Gln 520 525 530 atg gtt atg gat tgg cag gtt tcc ctc cgg agc ata gag ctt gga ggg 1728 Met Val Met Asp Trp Gln Val Ser Leu Arg Ser Ile Glu Leu Gly Gly 535 540 545 aag agc cgt gga ttc atc atg cac cgc cgg gtt gtg gga atg ctc caa 1776 Lys Ser Arg Gly Phe Ile Met His Arg Arg Val Val Gly Met Leu Gln 550 555 560 gat cat cgg gat ccg gga aga cac ccg aga tct ccc agg ctg ttc acg 1824 Asp His Arg Asp Pro Gly Arg His Pro Arg Ser Pro Arg Leu Phe Thr 565 570 575 580 ctg ctc acg ctg aaa tca acg aag ctg gtc gtg ccc ccg agg ctg atg 1872 Leu Leu Thr Leu Lys Ser Thr Lys Leu Val Val Pro Pro Arg Leu Met 585 590 595 cgc aac aaa ata act tca aca aag atc aac aaa gcg cct tct atg aaa 1920 Arg Asn Lys Ile Thr Ser Thr Lys Ile Asn Lys Ala Pro Ser Met Lys 600 605 610 tct tga aca tgc cta act taa acg aag cgc aac gta acg gct tca ttc 1968 Ser Thr Cys Leu Thr Thr Lys Arg Asn Val Thr Ala Ser Phe 615 620 625 aaa gtc tta aag acg acc caa gcc aaa gca cta acg ttt tag gtg aag 2016 Lys Val Leu Lys Thr Thr Gln Ala Lys Ala Leu Thr Phe Val Lys 630 635 640 cta aaa aat taa acg aat ctc aag cac cca aac ccg agg ctg atg cgc 2064 Leu Lys Asn Thr Asn Leu Lys His Pro Asn Pro Arg Leu Met Arg 645 650 655 aac aaa ata act tca aca aag atc aac aaa gcg cct tct atg aaa tct 2112 Asn Lys Ile Thr Ser Thr Lys Ile Asn Lys Ala Pro Ser Met Lys Ser 660 665 670 tga aca tgc cta act taa acg aag cgc aac gta acg gct tca ttc aaa 2160 Thr Cys Leu Thr Thr Lys Arg Asn Val Thr Ala Ser Phe Lys 675 680 685 gtc tta aag acg acc caa gcc aaa gca cta acg ttt tag gtg aag cta 2208 Val Leu Lys Thr Thr Gln Ala Lys Ala Leu Thr Phe Val Lys Leu 690 695 700 aaa aat taa acg aat ctc aag cac cca aac ccg agg tag cag gtc aga 2256 Lys Asn Thr Asn Leu Lys His Pro Asn Pro Arg Gln Val Arg 705 710 715 att agc ttg ctg att gat tga ccg gat cga tcc ggc tct aga att aat 2304 Ile Ser Leu Leu Ile Asp Pro Asp Arg Ser Gly Ser Arg Ile Asn 720 725 730 tca cct cga aag caa gct gat aaa ccg ata caa tta aag gct cct ttt 2352 Ser Pro Arg Lys Gln Ala Asp Lys Pro Ile Gln Leu Lys Ala Pro Phe 735 740 745 gga gcc ttt ttt ttt gga gat ttt caa cgt gaa aaa att att att cgc 2400 Gly Ala Phe Phe Phe Gly Asp Phe Gln Arg Glu Lys Ile Ile Ile Arg 750 755 760 aat tca agc taa ttc acc tag aaa gca agc tga taa acc gat aca att 2448 Asn Ser Ser Phe Thr Lys Ala Ser Thr Asp Thr Ile 765 770 aaa ggc tcc ttt tgg agc ctt ttt ttt tgg aga ttt tca acg tga aaa 2496 Lys Gly Ser Phe Trp Ser Leu Phe Phe Trp Arg Phe Ser Thr Lys 775 780 785 aat tat tat tcg caa ttc aag ctc tgc ctc gcg cgt ttc ggt gat gac 2544 Asn Tyr Tyr Ser Gln Phe Lys Leu Cys Leu Ala Arg Phe Gly Asp Asp 790 795 800 805 ggt gaa aac ctc tga cac atg cag ctc ccg gag acg gtc aca gct tgt 2592 Gly Glu Asn Leu His Met Gln Leu Pro Glu Thr Val Thr Ala Cys 810 815 820 ctg taa gcg gat gcc ggg agc aga caa gcc cgt cag ggc gcg tca gcg 2640 Leu Ala Asp Ala Gly Ser Arg Gln Ala Arg Gln Gly Ala Ser Ala 825 830 835 ggt gtt ggc ggg tgt cgg ggc gca gcc atg acc cag tca cgt agc gat 2688 Gly Val Gly Gly Cys Arg Gly Ala Ala Met Thr Gln Ser Arg Ser Asp 840 845 850 agc gga gtg tat gtg tct caa aat ctc tga tgt tac att gca caa gat 2736 Ser Gly Val Tyr Val Ser Gln Asn Leu Cys Tyr Ile Ala Gln Asp 855 860 865 aaa aat ata tca tca tga aca ata aaa ctg tct gct tac ata aac agt 2784 Lys Asn Ile Ser Ser Thr Ile Lys Leu Ser Ala Tyr Ile Asn Ser 870 875 880 aat aca agg ggt gtt atg agc cat att caa cgg gaa acg tct tgc tcg 2832 Asn Thr Arg Gly Val Met Ser His Ile Gln Arg Glu Thr Ser Cys Ser 885 890 895 agg ccg cga tta aat tcc aac atg gat gct gat tta tat ggg tat aaa 2880 Arg Pro Arg Leu Asn Ser Asn Met Asp Ala Asp Leu Tyr Gly Tyr Lys 900 905 910 tgg gct cgc gat aat gtc ggg caa tca ggt gcg aca atc tat cga ttg 2928 Trp Ala Arg Asp Asn Val Gly Gln Ser Gly Ala Thr Ile Tyr Arg Leu 915 920 925 tat ggg aag ccc gat gcg cca gag ttg ttt ctg aaa cat ggc aaa ggt 2976 Tyr Gly Lys Pro Asp Ala Pro Glu Leu Phe Leu Lys His Gly Lys Gly 930 935 940 945 agc gtt gcc aat gat gtt aca gat gag atg gtc aga cta aac tgg ctg 3024 Ser Val Ala Asn Asp Val Thr Asp Glu Met Val Arg Leu Asn Trp Leu 950 955 960 acg gaa ttt atg cct ctt ccg acc atc aag cat ttt atc cgt act cct 3072 Thr Glu Phe Met Pro Leu Pro Thr Ile Lys His Phe Ile Arg Thr Pro 965 970 975 gat gat gca tgg tta ctc acc act gcg atc ccc ggg aaa aca gca ttc 3120 Asp Asp Ala Trp Leu Leu Thr Thr Ala Ile Pro Gly Lys Thr Ala Phe 980 985 990 cag gta tta gaa gaa tat cct gat tca ggt gaa aat att gtt gat gcg 3168 Gln Val Leu Glu Glu Tyr Pro Asp Ser Gly Glu Asn Ile Val Asp Ala 995 1000 1005 ctg gca gtg ttc ctg cgc cgg ttg cat tcg att cct gtt tgt aat 3213 Leu Ala Val Phe Leu Arg Arg Leu His Ser Ile Pro Val Cys Asn 1010 1015 1020 tgt cct ttt aac agc gat cgc gta ttt cgt ctc gct cag gcg caa 3258 Cys Pro Phe Asn Ser Asp Arg Val Phe Arg Leu Ala Gln Ala Gln 1025 1030 1035 tca cga atg aat aac ggt ttg gtt gat gcg agt gat ttt gag acg 3303 Ser Arg Met Asn Asn Gly Leu Val Asp Ala Ser Asp Phe Glu Thr 1040 1045 1050 agc gta atg gct ggc ctg ttg aac aag tct gga aag aaa tgc ata 3348 Ser Val Met Ala Gly Leu Leu Asn Lys Ser Gly Lys Lys Cys Ile 1055 1060 1065 aac ttt tgc cat tct cac cgg att cag tcg tca ctc atg gtg att 3393 Asn Phe Cys His Ser His Arg Ile Gln Ser Ser Leu Met Val Ile 1070 1075 1080 tct cac ttg ata acc tta ttt ttg acg agg gga aat taa tag gtt 3438 Ser His Leu Ile Thr Leu Phe Leu Thr Arg Gly Asn Val 1085 1090 1095 gta ttg atg ttg gac gag tcg gaa tcg cag acc gat acc agg atc 3483 Val Leu Met Leu Asp Glu Ser Glu Ser Gln Thr Asp Thr Arg Ile 1100 1105 1110 ttg cca tcc tat gga act gcc tcg gtg agt ttt ctc ctt cat tac 3528 Leu Pro Ser Tyr Gly Thr Ala Ser Val Ser Phe Leu Leu His Tyr 1115 1120 1125 aga aac ggc ttt ttc aaa aat atg gta ttg ata atc ctg ata tga 3573 Arg Asn Gly Phe Phe Lys Asn Met Val Leu Ile Ile Leu Ile 1130 1135 1140 ata aat tgc agt ttc att tga tgc tcg atg agt ttt tct aat cag 3618 Ile Asn Cys Ser Phe Ile Cys Ser Met Ser Phe Ser Asn Gln 1145 1150 1155 aat tgg tta att ggt tgt aac act ggc aga gca tta cgc tga ctt 3663 Asn Trp Leu Ile Gly Cys Asn Thr Gly Arg Ala Leu Arg Leu 1160 1165 gac ggg acg gcg gct ttg ttg aat aaa tcg aac ttt tgc tga gtt 3708 Asp Gly Thr Ala Ala Leu Leu Asn Lys Ser Asn Phe Cys Val 1170 1175 1180 gaa gga tca gat cac gca tct tcc cga caa cgc aga ccg ttc cgt 3753 Glu Gly Ser Asp His Ala Ser Ser Arg Gln Arg Arg Pro Phe Arg 1185 1190 1195 ggc aaa gca aaa gtt caa aat cac caa ctg gtc cgg atc gat ccg 3798 Gly Lys Ala Lys Val Gln Asn His Gln Leu Val Arg Ile Asp Pro 1200 1205 1210 4 1213 PRT Artificial Sequence Description of Artificial Sequence Synthetic fusion protein 4 Val Pro His Leu Ser Ile Gly Asn Cys Gln Thr Lys Phe Thr His Ile 1 5 10 15 Tyr Phe Arg Leu Ile Asn Phe Ile Phe Asn Leu Lys Gly Ser Arg Arg 20 25 30 Ser Phe Leu Ile Ile Ser Pro Lys Ser Leu Asn Val Ser Phe Arg Ser 35 40 45 Thr Glu Arg Gln Thr Pro Lys Arg Ser Lys Asp Leu Leu Glu Ile Leu 50 55 60 Phe Phe Cys Ala Ser Ala Ala Cys Lys Gln Lys Asn His Arg Tyr Gln 65 70 75 80 Arg Trp Phe Val Cys Arg Ile Lys Ser Tyr Gln Leu Phe Phe Arg Arg 85 90 95 Leu Ala Ser Ala Glu Arg Arg Tyr Gln Ile Leu Ser Phe Cys Ser Arg 100 105 110 Ser Ala Thr Thr Ser Arg Thr Leu His Arg Leu His Thr Ser Leu Cys 115 120 125 Ser Cys Tyr Gln Trp Leu Leu Pro Val Ala Ile Ser Arg Val Leu Pro 130 135 140 Gly Trp Thr Gln Asp Asp Ser Tyr Arg Ile Arg Arg Ser Gly Arg Ala 145 150 155 160 Glu Arg Gly Val Arg Ala His Ser Pro Ala Trp Ser Glu Arg Pro Thr 165 170 175 Pro Asn Asp Thr Tyr Ser Val Ser Tyr Glu Lys Ala Pro Arg Phe Pro 180 185 190 Lys Gly Glu Arg Arg Thr Gly Ile Arg Ala Ala Gly Ser Glu Gln Glu 195 200 205 Ser Ala Arg Gly Ser Phe Gln Gly Glu Thr Pro Gly Ile Phe Ile Val 210 215 220 Leu Ser Gly Phe Ala Thr Ser Asp Leu Ser Val Asp Phe Cys Asp Ala 225 230 235 240 Arg Gln Gly Gly Gly Ala Tyr Gly Lys Thr Pro Ala Thr Arg Pro Phe 245 250 255 Tyr Gly Ser Trp Pro Phe Ala Gly Leu Leu Leu Thr Cys Ser Phe Leu 260 265 270 Arg Tyr Pro Leu Ile Leu Trp Ile Thr Val Leu Pro Pro Leu Ser Glu 275 280 285 Leu Ile Pro Leu Ala Ala Ala Glu Arg Pro Ser Ala Ala Ser Gln Ala 290 295 300 Arg Lys Arg Lys Ser Ala Gln Tyr Ala Asn Arg Leu Ser Pro Arg Val 305 310 315 320 Gly Arg Phe Ile Asn Ala Glu Arg Pro Pro Gln Gly Ala Leu Pro Phe 325 330 335 Trp Ile Met Phe Phe Ala Pro Thr Ser Arg Phe Trp Gln Ile Phe Asn 340 345 350 Glu Leu Leu Thr Ile Asn His Arg Thr Ser Leu Val Arg Lys Phe Thr 355 360 365 Lys Gly Tyr Arg Gln Lys Gln Phe Ser Tyr Lys Leu Leu Met Met Ile 370 375 380 Ser Tyr Ile Gly Gln Ile Leu Asp Leu Leu Met Lys Ser Ser Gln Val 385 390 395 400 Val Leu Cys Gln Glu Asp Arg Val Ser Thr Leu Thr Glu Val Leu Lys 405 410 415 Ile Ser Thr Phe Met Ile Met Gln Glu Glu Leu Arg Arg Asp Leu Leu 420 425 430 Gly Thr Met Met Asp Met Phe Pro Pro Gln Leu Val Glu Val Pro Thr 435 440 445 Trp Val Lys Leu Tyr Cys Leu Val Ile Leu Leu Ile Ile Tyr Met Leu 450 455 460 Pro Leu His Pro Thr Cys Leu Thr Leu Met Met Tyr Gly His Thr Val 465 470 475 480 Leu Ile Gln Met Asn Lys Lys Phe Leu Leu Val Gly Phe His Thr Pro 485 490 495 Lys Tyr Met Asp Gly Ile Glu Phe Ile Leu Gly Cys Leu Met Asn Asn 500 505 510 Tyr Ile Val Ile Gly Ala Thr Glu Ile Asp Ile Thr Val Thr Ile Leu 515 520 525 Leu Gln Gln Gln Met Val Met Asp Trp Gln Val Ser Leu Arg Ser Ile 530 535 540 Glu Leu Gly Gly Lys Ser Arg Gly Phe Ile Met His Arg Arg Val Val 545 550 555 560 Gly Met Leu Gln Asp His Arg Asp Pro Gly Arg His Pro Arg Ser Pro 565 570 575 Arg Leu Phe Thr Leu Leu Thr Leu Lys Ser Thr Lys Leu Val Val Pro 580 585 590 Pro Arg Leu Met Arg Asn Lys Ile Thr Ser Thr Lys Ile Asn Lys Ala 595 600 605 Pro Ser Met Lys Ser Thr Cys Leu Thr Thr Lys Arg Asn Val Thr Ala 610 615 620 Ser Phe Lys Val Leu Lys Thr Thr Gln Ala Lys Ala Leu Thr Phe Val 625 630 635 640 Lys Leu Lys Asn Thr Asn Leu Lys His Pro Asn Pro Arg Leu Met Arg 645 650 655 Asn Lys Ile Thr Ser Thr Lys Ile Asn Lys Ala Pro Ser Met Lys Ser 660 665 670 Thr Cys Leu Thr Thr Lys Arg Asn Val Thr Ala Ser Phe Lys Val Leu 675 680 685 Lys Thr Thr Gln Ala Lys Ala Leu Thr Phe Val Lys Leu Lys Asn Thr 690 695 700 Asn Leu Lys His Pro Asn Pro Arg Gln Val Arg Ile Ser Leu Leu Ile 705 710 715 720 Asp Pro Asp Arg Ser Gly Ser Arg Ile Asn Ser Pro Arg Lys Gln Ala 725 730 735 Asp Lys Pro Ile Gln Leu Lys Ala Pro Phe Gly Ala Phe Phe Phe Gly 740 745 750 Asp Phe Gln Arg Glu Lys Ile Ile Ile Arg Asn Ser Ser Phe Thr Lys 755 760 765 Ala Ser Thr Asp Thr Ile Lys Gly Ser Phe Trp Ser Leu Phe Phe Trp 770 775 780 Arg Phe Ser Thr Lys Asn Tyr Tyr Ser Gln Phe Lys Leu Cys Leu Ala 785 790 795 800 Arg Phe Gly Asp Asp Gly Glu Asn Leu His Met Gln Leu Pro Glu Thr 805 810 815 Val Thr Ala Cys Leu Ala Asp Ala Gly Ser Arg Gln Ala Arg Gln Gly 820 825 830 Ala Ser Ala Gly Val Gly Gly Cys Arg Gly Ala Ala Met Thr Gln Ser 835 840 845 Arg Ser Asp Ser Gly Val Tyr Val Ser Gln Asn Leu Cys Tyr Ile Ala 850 855 860 Gln Asp Lys Asn Ile Ser Ser Thr Ile Lys Leu Ser Ala Tyr Ile Asn 865 870 875 880 Ser Asn Thr Arg Gly Val Met Ser His Ile Gln Arg Glu Thr Ser Cys 885 890 895 Ser Arg Pro Arg Leu Asn Ser Asn Met Asp Ala Asp Leu Tyr Gly Tyr 900 905 910 Lys Trp Ala Arg Asp Asn Val Gly Gln Ser Gly Ala Thr Ile Tyr Arg 915 920 925 Leu Tyr Gly Lys Pro Asp Ala Pro Glu Leu Phe Leu Lys His Gly Lys 930 935 940 Gly Ser Val Ala Asn Asp Val Thr Asp Glu Met Val Arg Leu Asn Trp 945 950 955 960 Leu Thr Glu Phe Met Pro Leu Pro Thr Ile Lys His Phe Ile Arg Thr 965 970 975 Pro Asp Asp Ala Trp Leu Leu Thr Thr Ala Ile Pro Gly Lys Thr Ala 980 985 990 Phe Gln Val Leu Glu Glu Tyr Pro Asp Ser Gly Glu Asn Ile Val Asp 995 1000 1005 Ala Leu Ala Val Phe Leu Arg Arg Leu His Ser Ile Pro Val Cys Asn 1010 1015 1020 Cys Pro Phe Asn Ser Asp Arg Val Phe Arg Leu Ala Gln Ala Gln Ser 1025 1030 1035 1040 Arg Met Asn Asn Gly Leu Val Asp Ala Ser Asp Phe Glu Thr Ser Val 1045 1050 1055 Met Ala Gly Leu Leu Asn Lys Ser Gly Lys Lys Cys Ile Asn Phe Cys 1060 1065 1070 His Ser His Arg Ile Gln Ser Ser Leu Met Val Ile Ser His Leu Ile 1075 1080 1085 Thr Leu Phe Leu Thr Arg Gly Asn Val Val Leu Met Leu Asp Glu Ser 1090 1095 1100 Glu Ser Gln Thr Asp Thr Arg Ile Leu Pro Ser Tyr Gly Thr Ala Ser 1105 1110 1115 1120 Val Ser Phe Leu Leu His Tyr Arg Asn Gly Phe Phe Lys Asn Met Val 1125 1130 1135 Leu Ile Ile Leu Ile Ile Asn Cys Ser Phe Ile Cys Ser Met Ser Phe 1140 1145 1150 Ser Asn Gln Asn Trp Leu Ile Gly Cys Asn Thr Gly Arg Ala Leu Arg 1155 1160 1165 Leu Asp Gly Thr Ala Ala Leu Leu Asn Lys Ser Asn Phe Cys Val Glu 1170 1175 1180 Gly Ser Asp His Ala Ser Ser Arg Gln Arg Arg Pro Phe Arg Gly Lys 1185 1190 1195 1200 Ala Lys Val Gln Asn His Gln Leu Val Arg Ile Asp Pro 1205 1210 5 1176 PRT Artificial Sequence Description of Artificial Sequence Synthetic fusion protein 5 Cys Leu Thr Asp Ala Leu Val Thr Val Arg Pro Ser Leu Leu Ile Tyr 1 5 10 15 Thr Leu Asp Phe Lys Thr Ser Phe Leu Ile Lys Asp Leu Gly Glu Asp 20 25 30 Pro Phe Ser His Asp Gln Asn Pro Leu Thr Val Phe Val Pro Leu Ser 35 40 45 Val Arg Pro Arg Arg Lys Asp Gln Arg Ile Phe Leu Arg Ser Phe Phe 50 55 60 Ser Ala Arg Asn Leu Leu Leu Ala Asn Lys Lys Thr Thr Ala Thr Ser 65 70 75 80 Gly Gly Leu Phe Ala Gly Ser Arg Ala Thr Asn Ser Phe Ser Glu Gly 85 90 95 Asn Trp Leu Gln Gln Ser Ala Asp Thr Lys Tyr Cys Pro Ser Ser Val 100 105 110 Ala Val Val Arg Pro Pro Leu Gln Glu Leu Cys Ser Thr Ala Tyr Ile 115 120 125 Pro Arg Ser Ala Asn Pro Val Thr Ser Gly Cys Cys Gln Trp Arg Val 130 135 140 Val Ser Tyr Arg Val Gly Leu Lys Thr Ile Val Thr Gly Gly Ala Ala 145 150 155 160 Val Gly Leu Asn Gly Gly Phe Val His Thr Ala Gln Leu Gly Ala Asn 165 170 175 Asp Leu His Arg Thr Glu Ile Pro Thr Ala Ala Met Arg Lys Arg His 180 185 190 Ala Ser Arg Arg Glu Lys Gly Gly Gln Val Ser Gly Lys Arg Gln Gly 195 200 205 Arg Asn Arg Arg Ala His Glu Gly Ala Ser Arg Gly Lys Arg Leu Val 210 215 220 Ser Leu Ser Cys Arg Val Ser Pro Pro Leu Thr Ala Ser Ile Phe Val 225 230 235 240 Met Leu Val Arg Gly Ala Glu Pro Met Glu Lys Arg Gln Gln Arg Gly 245 250 255 Leu Phe Thr Val Pro Gly Leu Leu Leu Ala Phe Cys Ser His Val Leu 260 265 270 Ser Cys Val Ile Pro Phe Cys Gly Pro Tyr Tyr Arg Leu Val Ser Tyr 275 280 285 Arg Ser Pro Gln Pro Asn Asp Arg Ala Gln Arg Val Ser Glu Arg Gly 290 295 300 Ser Gly Arg Ala Pro Asn Thr Gln Thr Ala Ser Pro Arg Ala Leu Ala 305 310 315 320 Asp Ser Leu Met Gln Ser Gly Arg Leu Lys Ala His Ser Arg Ser Gly 325 330 335 Cys Phe Leu Arg Arg His His Asn Gly Ser Gly Lys Tyr Ser Glu Met 340 345 350 Ser Cys Gln Leu Ile Ile Glu Leu Val Asn Tyr Ala Ser Ser Arg Lys 355 360 365 Lys Gly Ile Asp Asn Glu Ser Asn Phe Arg Thr Glu Ser Phe Val Ile 370 375 380 Ser Gly Arg Phe Thr Ser Asn Lys Ala Val Arg Trp Ser Tyr Ala Lys 385 390 395 400 Arg Thr Glu Val Leu Pro Arg Tyr Ser Asn Glu Tyr Gln Pro Leu Ser 405 410 415 Cys Lys Arg Asn Ser Asp Gly Ile Cys Ala Arg Trp Ile Cys Phe His 420 425 430 Leu Asn Phe Glu Lys Cys Pro Leu Ser Gly Ser Asn Tyr Ile Val Trp 435 440 445 Ser Phe Tyr Leu Leu Tyr Ile Cys Tyr Ser His Cys Thr Gln His Val 450 455 460 Arg Cys Ile Arg Gly Ile Gln Ser Ser Ser Arg Thr Arg Ser Phe Cys 465 470 475 480 Phe Arg Trp Asp Ser Ile Leu Pro Asn Ile Trp Met Val Ser Ser Ser 485 490 495 Phe Trp Gly Ala Thr Ile Thr Ser Gly Leu Gln Arg Ile Leu Gln Leu 500 505 510 Arg Tyr Cys Ser Ser Ser Arg Trp Leu Trp Ile Gly Arg Phe Pro Ser 515 520 525 Gly Ala Ser Leu Glu Gly Arg Ala Val Asp Ser Ser Cys Thr Ala Gly 530 535 540 Leu Trp Glu Cys Ser Lys Ile Ile Gly Ile Arg Glu Asp Thr Arg Asp 545 550 555 560 Leu Pro Gly Cys Ser Arg Cys Ser Arg Asn Gln Arg Ser Trp Ser Cys 565 570 575 Pro Arg Gly Cys Ala Thr Lys Leu Gln Gln Arg Ser Thr Lys Arg Leu 580 585 590 Leu Asn Leu Glu His Ala Leu Lys Arg Ser Ala Thr Arg Leu His Ser 595 600 605 Lys Ser Arg Arg Pro Lys Pro Lys His Arg Phe Arg Ser Lys Ile Lys 610 615 620 Arg Ile Ser Ser Thr Gln Thr Arg Gly Cys Ala Thr Lys Leu Gln Gln 625 630 635 640 Arg Ser Thr Lys Arg Leu Leu Asn Leu Glu His Ala Leu Lys Arg Ser 645 650 655 Ala Thr Arg Leu His Ser Lys Ser Arg Arg Pro Lys Pro Lys His Arg 660 665 670 Phe Arg Ser Lys Ile Lys Arg Ile Ser Ser Thr Gln Thr Arg Gly Ser 675 680 685 Arg Ser Glu Leu Ala Cys Leu Ile Asp Arg Ile Asp Pro Ala Leu Glu 690 695 700 Leu Ile His Leu Glu Ser Lys Leu Ile Asn Arg Tyr Asn Arg Leu Leu 705 710 715 720 Leu Glu Pro Phe Phe Leu Glu Ile Phe Asn Val Lys Lys Leu Leu Phe 725 730 735 Ala Ile Gln Ala Asn Ser Pro Arg Lys Gln Ala Asp Lys Pro Ile Gln 740 745 750 Leu Lys Ala Pro Phe Gly Ala Phe Phe Phe Gly Asp Phe Gln Arg Glu 755 760 765 Lys Ile Ile Ile Arg Asn Ser Ser Ser Ala Ser Arg Val Ser Val Met 770 775 780 Thr Val Lys Thr Ser Asp Thr Cys Ser Ser Arg Arg Arg Ser Gln Leu 785 790 795 800 Val Cys Lys Arg Met Pro Gly Ala Asp Lys Pro Val Arg Ala Arg Gln 805 810 815 Arg Val Leu Ala Gly Val Gly Ala Gln Pro Pro Ser His Val Ala Ile 820 825 830 Ala Glu Cys Met Cys Leu Lys Ile Ser Asp Val Thr Leu His Lys Ile 835 840 845 Lys Ile Tyr His His Glu Gln Asn Cys Leu Leu Thr Thr Val Ile Gln 850 855 860 Gly Val Leu Ala Ile Phe Asn Gly Lys Arg Leu Ala Arg Gly Arg Asp 865 870 875 880 Ile Pro Thr Trp Met Leu Ile Tyr Met Gly Ile Asn Gly Leu Ala Ile 885 890 895 Met Ser Gly Asn Gln Val Arg Gln Ser Ile Asp Cys Met Gly Ser Pro 900 905 910 Met Arg Gln Ser Cys Phe Asn Met Ala Lys Val Ala Leu Pro Met Met 915 920 925 Leu Gln Met Arg Trp Ser Asp Thr Gly Arg Asn Leu Cys Leu Phe Arg 930 935 940 Pro Ser Ser Ile Leu Ser Val Leu Leu Met Met His Gly Tyr Ser Pro 945 950 955 960 Leu Arg Ser Pro Gly Lys Gln His Ser Arg Tyr Lys Asn Ile Leu Ile 965 970 975 Gln Val Lys Ile Leu Leu Met Arg Trp Gln Cys Ser Cys Ala Gly Cys 980 985 990 Ile Arg Phe Leu Phe Val Ile Val Leu Leu Thr Ala Ile Ala Tyr Phe 995 1000 1005 Val Ser Leu Arg Arg Asn His Glu Ile Thr Val Trp Leu Met Arg Val 1010 1015 1020 Ile Leu Arg Arg Ala Trp Leu Ala Cys Thr Ser Leu Glu Arg Asn Ala 1025 1030 1035 1040 Thr Phe Ala Ile Leu Thr Gly Phe Ser Arg His Ser Trp Phe Leu Thr 1045 1050 1055 Pro Tyr Phe Arg Gly Glu Ile Asn Arg Leu Tyr Cys Trp Thr Ser Arg 1060 1065 1070 Asn Arg Arg Pro Ile Pro Gly Ser Cys His Pro Met Glu Leu Pro Arg 1075 1080 1085 Val Phe Ser Phe Ile Thr Glu Thr Ala Phe Ser Lys Ile Trp Tyr Ser 1090 1095 1100 Tyr Glu Ile Ala Val Ser Phe Asp Ala Arg Val Phe Leu Ile Arg Ile 1105 1110 1115 1120 Gly Leu Val Val Thr Leu Ala Glu His Tyr Ala Asp Leu Thr Gly Arg 1125 1130 1135 Arg Leu Cys Ile Asn Arg Thr Phe Ala Glu Leu Lys Asp Gln Ile Thr 1140 1145 1150 His Leu Pro Asp Asn Ala Asp Arg Ser Val Ala Lys Gln Lys Phe Lys 1155 1160 1165 Ile Thr Asn Trp Ser Gly Ser Ile 1170 1175 6 1209 PRT Artificial Sequence Description of Artificial Sequence Synthetic fusion protein 6 Ala Ser Leu Ile Lys His Trp Leu Ser Asp Gln Val Tyr Ser Tyr Ile 1 5 10 15 Leu Ile Asp Leu Lys Leu His Phe Phe Lys Arg Ile Val Lys Ile Leu 20 25 30 Phe Asp Asn Leu Met Thr Lys Ile Pro Arg Glu Phe Ser Phe His Ala 35 40 45 Ser Asp Pro Val Glu Lys Ile Lys Gly Ser Ser Asp Pro Phe Phe Leu 50 55 60 Arg Val Ile Cys Cys Leu Gln Thr Lys Lys Pro Pro Leu Pro Ala Val 65 70 75 80 Val Cys Leu Pro Asp Gln Glu Leu Pro Thr Leu Phe Pro Lys Val Thr 85 90 95 Gly Phe Ser Arg Ala Gln Ile Pro Asn Thr Val Leu Leu Val Pro Leu 100 105 110 Gly His His Phe Lys Asn Ser Val Ala Pro Pro Thr Tyr Leu Ala Leu 115 120 125 Leu Ile Leu Leu Pro Val Ala Ala Ala Ser Gly Asp Lys Ser Cys Leu 130 135 140 Thr Gly Leu Asp Ser Arg Arg Leu Pro Asp Lys Ala Gln Arg Ser Gly 145 150 155 160 Thr Gly Gly Ser Cys Thr Gln Pro Ser Leu Glu Arg Thr Thr Tyr Thr 165 170 175 Glu Leu Arg Tyr Leu Gln Arg Glu Leu Glu Ser Ala Thr Leu Pro Glu 180 185 190 Gly Arg Lys Ala Asp Arg Tyr Pro Val Ser Gly Arg Val Gly Thr Gly 195 200 205 Glu Arg Thr Arg Glu Leu Pro Gly Gly Asn Ala Trp Tyr Leu Tyr Ser 210 215 220 Pro Val Gly Phe Arg His Leu Leu Glu Arg Arg Phe Leu Cys Ser Ser 225 230 235 240 Gly Gly Arg Ser Leu Trp Lys Asn Ala Ser Asn Ala Ala Phe Leu Arg 245 250 255 Phe Leu Ala Phe Cys Trp Pro Phe Ala His Met Phe Phe Pro Ala Leu 260 265 270 Ser Pro Asp Ser Val Asp Asn Arg Ile Thr Ala Phe Glu Ala Asp Thr 275 280 285 Ala Arg Arg Ser Arg Thr Thr Glu Arg Ser Glu Ser Val Ser Glu Glu 290 295 300 Ala Glu Glu Arg Pro Ile Arg Lys Pro Pro Leu Pro Ala Arg Trp Pro 305 310 315 320 Ile His Cys Arg Ala Ala Ala Ser Arg Arg Thr Pro Val Leu Asp Asn 325 330 335 Val Phe Cys Ala Asp Ile Ile Thr Val Leu Ala Asn Ile Leu Lys Ala 340 345 350 Val Asp Asn Ser Ser Asn Leu Thr Ser Thr Gln Val His Val Lys Arg 355 360 365 Val Ser Thr Met Lys Ala Ile Phe Val Leu Lys Ala Ser Asn Asp Asp 370 375 380 Lys Leu Tyr Arg Ala Asp Ser Arg Pro Pro Asp Glu Ile Lys Gln Ser 385 390 395 400 Gly Gly Leu Met Pro Arg Gly Gln Ser Glu Tyr Phe Asp Arg Gly Thr 405 410 415 Gln Met Asn Ile Asn Leu Tyr Asp His Ala Arg Gly Thr Gln Thr Gly 420 425 430 Phe Val Arg His Asp Asp Gly Tyr Val Ser Thr Ser Ile Ser Leu Arg 435 440 445 Ser Ala His Leu Val Gly Gln Thr Ile Leu Ser Gly His Ser Thr Tyr 450 455 460 Tyr Ile Tyr Val Ile Ala Thr Ala Pro Asn Met Phe Asn Val Asn Asp 465 470 475 480 Val Leu Gly Ala Tyr Ser Pro His Pro Asp Glu Gln Glu Val Ser Ala 485 490 495 Leu Gly Gly Ile Pro Tyr Ser Gln Ile Tyr Gly Trp Tyr Arg Val His 500 505 510 Phe Gly Val Leu Asp Glu Gln Leu His Arg Asn Arg Gly Tyr Arg Asp 515 520 525 Arg Tyr Tyr Ser Asn Leu Asp Ile Ala Pro Ala Ala Asp Gly Tyr Gly 530 535 540 Leu Ala Gly Phe Pro Pro Glu His Arg Ala Trp Arg Glu Glu Pro Trp 545 550 555 560 Ile His His Ala Pro Pro Gly Cys Gly Asn Ala Pro Arg Ser Ser Gly 565 570 575 Ser Gly Lys Thr Pro Glu Ile Ser Gln Ala Val His Ala Ala His Ala 580 585 590 Glu Ile Asn Glu Ala Gly Arg Ala Pro Glu Ala Asp Ala Gln Gln Asn 595 600 605 Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met 610 615 620 Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys 625 630 635 640 Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu 645 650 655 Asn Glu Ser Gln Ala Pro Lys Pro Glu Ala Asp Ala Gln Gln Asn Asn 660 665 670 Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro 675 680 685 Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp 690 695 700 Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn 705 710 715 720 Glu Ser Gln Ala Pro Lys Pro Glu Val Ala Gly Gln Asn Leu Ala Asp 725 730 735 Leu Thr Gly Ser Ile Arg Leu Asn Phe Thr Ser Lys Ala Ser Thr Asp 740 745 750 Thr Ile Lys Gly Ser Phe Trp Ser Leu Phe Phe Trp Arg Phe Ser Thr 755 760 765 Lys Asn Tyr Tyr Ser Gln Phe Lys Leu Ile His Leu Glu Ser Lys Leu 770 775 780 Ile Asn Arg Tyr Asn Arg Leu Leu Leu Glu Pro Phe Phe Leu Glu Ile 785 790 795 800 Phe Asn Val Lys Lys Leu Leu Phe Ala Ile Gln Ala Leu Pro Arg Ala 805 810 815 Phe Arg Arg Lys Pro Leu Thr His Ala Ala Pro Gly Asp Gly His Ser 820 825 830 Leu Ser Val Ser Gly Cys Arg Glu Gln Thr Ser Pro Ser Gly Arg Val 835 840 845 Ser Gly Cys Trp Arg Val Ser Gly Arg Ser His Asp Pro Val Thr Arg 850 855 860 Arg Ser Val Cys Val Ser Lys Ser Leu Met Leu His Cys Thr Arg Lys 865 870 875 880 Tyr Ile Ile Met Asn Asn Lys Thr Val Cys Leu His Lys Gln Tyr Lys 885 890 895 Gly Cys Tyr Glu Pro Tyr Ser Thr Gly Asn Val Leu Leu Glu Ala Ala 900 905 910 Ile Lys Phe Gln His Gly Cys Phe Ile Trp Val Met Gly Ser Arg Cys 915 920 925 Arg Ala Ile Arg Cys Asp Asn Leu Ser Ile Val Trp Glu Ala Arg Cys 930 935 940 Ala Arg Val Val Ser Glu Thr Trp Gln Arg Arg Cys Gln Cys Tyr Arg 945 950 955 960 Asp Gly Gln Thr Lys Leu Ala Asp Gly Ile Tyr Ala Ser Ser Asp His 965 970 975 Gln Ala Phe Tyr Pro Tyr Ser Cys Met Val Thr His His Cys Asp Pro 980 985 990 Arg Glu Asn Ser Ile Pro Gly Ile Arg Arg Ile Ser Phe Arg Lys Tyr 995 1000 1005 Cys Cys Ala Gly Ser Val Pro Ala Pro Val Ala Phe Asp Ser Cys Leu 1010 1015 1020 Leu Ser Phe Gln Arg Ser Arg Ile Ser Ser Arg Ser Gly Ala Ile Thr 1025 1030 1035 1040 Asn Glu Arg Phe Gly Cys Glu Phe Asp Glu Arg Asn Gly Trp Pro Val 1045 1050 1055 Glu Gln Val Trp Lys Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp 1060 1065 1070 Ser Val Val Thr His Gly Asp Phe Ser Leu Asp Asn Leu Ile Phe Asp 1075 1080 1085 Glu Gly Lys Leu Ile Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala 1090 1095 1100 Asp Arg Tyr Gln Asp Leu Ala Ile Leu Trp Asn Cys Leu Gly Glu Phe 1105 1110 1115 1120 Ser Pro Ser Leu Gln Lys Arg Leu Phe Gln Lys Tyr Gly Ile Asp Asn 1125 1130 1135 Pro Asp Met Asn Lys Leu Gln Phe His Leu Met Leu Asp Glu Phe Phe 1140 1145 1150 Ser Glu Leu Val Asn Trp Leu His Trp Gln Ser Ile Thr Leu Thr Arg 1155 1160 1165 Asp Gly Gly Phe Val Glu Ile Glu Leu Leu Leu Ser Arg Ile Arg Ser 1170 1175 1180 Arg Ile Phe Pro Thr Thr Gln Thr Val Pro Trp Gln Ser Lys Ser Ser 1185 1190 1195 1200 Lys Ser Pro Thr Gly Pro Asp Arg Ser 1205 

1. An immunogenic complex comprising at least one glycoside and at least one lipid, integrated into an iscom complex or matrix, and at least one antigen which antigen is integrated into the iscom complex or coupled on to or mixed with the iscom complex or iscom matrix complex, characterised in that it also comprises at least one enzyme.
 2. An immunogenic complex according to claim 1, characterised in that the enzyme confers enzymatic ADP-ribosylating activity.
 3. An immunogenic complex according to claim 1 and 2, characterised in that the enzyme is selected from Cholera toxin (CT), E. Coli beat labile enterotoxin (LT), Pertussis, Clostridia, Shigella ad Peudomonas toxins.
 4. An immunogenic complex according to claim 1-3, characterised in that the enzyme is selected from an A1 subunit of 4 bacterial enterotoxin wherein said enterotoxin is selected from the group consisting of cholera toxin (CT) and E. Coli heat labile enterotoxin (LT),
 5. An immunogenic complex according to claim 1-4, characterised in that it further comprises at least one peptide or protein, which specifically binds to a receptor expressed on a cell capable of antigen presentation, which cell expresses MHC Class I or Class II antigen when said antigen-presenting cell is selected from the group consisting of lymphocytes, macrophages, dendritic cells, Langerhans cells and epithelial cells.
 6. An immunogenic complex according to claims 1-5, characterised in that the enzyme is integrated into an iscom complex comprising at least at least one glycoside, at least one lipid and at least one antigen.
 7. An immunogenic complex according anyone of claim 1-5, characterised in that the peptide or protein which specifically binds to a receptor is integrated into an iscom complex comprising at least at least one glycoside, at least one lipid and at least one antigen.
 8. An immunogenic complex according to anyone of claims 1-7, characterised in that the enzyme coupled on to a iscom complex comprising at least at least one glycoside, at least one lipid and at least one antigen or on to a iscom matrix complex comprising at least at least one glycoside and at least one lipid.
 9. An immunogenic complex according to anyone of claims 5-8, characterised in that the peptide or protein, which specifically binds to a receptor is coupled on to a iscom complex comprising at least at least one glycoside, at least one lipid and at least one antigen or onto a iscom matrix complex comprising at least at least one glycoside and at least one lipid.
 10. An immunogenic complex according to anyone of claims 1-9, characterised in that the enzyme is mixed with a iscom complex comprising at least at least one glycoside, at least one lipid and at least one antigen or mixed with a iscom matrix complex comprising at least at least one glycoside and at least one lipid.
 11. An immunogenic complex according to anyone of claims 5-10, characterised in that the peptide or protein, which specifically binds to a receptor is mixed with a iscom complex comprising at least at least one glycoside, at least one lipid and at least one antigen or mixed with a iscom matrix complex comprising at least at least one glycoside and at least one lipid.
 12. An immunogenic complex according to anyone of claims 1-11, characterised in that said bacterial A1 enterotoxin subunit comprises the A1 subunit of cholera toxin.
 13. An immunogenic complex according to anyone of claims 5-12, characterised in That the antigen-presenting cell is a lymphocyte.
 14. An immunogenic complex according to anyone of claims 5-13, characterised in that the g-presenting cell is selected from the group consisting of macrophages, dendrii cells, Langerhans cells, and epithelial cells.
 15. An immunogenic complex according to anyone of claims 5-14 characterised in that the antigen-presenting cell is a B-lymphocyte.
 16. An immunogenic complex according to anyone of claims 5-15, characterised in that the peptide specifically binds to an Ig or Fc receptor expressed by said antigenpresenting cell.
 17. An immunogenic complex according to anyone of claims 5-16, characterised in that the peptide which specifically-binds said antigen-presenting cell is selected from the group consisting of protein A, a fragment thereof and multiuners thereof
 18. An immunogenic complex according to anyone of claims 1-17, characterised in that the enzyme and the peptide or protein which specifically binds to a receptor is bound together into a fusion protein, which is integrated into an is co=complex or couled on to or mixed with an iscom complex or iscom matrix complex.
 19. An immunogenic complex according to anyone of claims 1-18, coharactrised in that the enzyme, the peptide or prote which specifically binds to a receptor and a aigen is bound together into a fusion protein, which is integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscorn matr complex
 20. An immunogenic complex according to anyone of claims 18-19, characterised in Iat the asion protein comprises the Al subunit of cholera toxin fued to one or more copies of protein A or a fragment Thereof
 21. An immunogenic complex according t claim 20, characterised in that the fragment of protein A comprises the D region of said protein A.
 22. An immunogenic complex according to claims 1-21, characterised in that it further comprises other immunomodulatory compounds or targetig agents,
 23. An immunogenic composition according to claims 122, characterised in that it further comprises one or more excipients that are acceptable in pharmaceutical or vete products, whereby complexes and components to be mixed therewith may be placed in separate compartments. 